Method for detoxifying phosphonate herbicides

ABSTRACT

The invention relates in general to methods for detoxifying phosphonate herbicides. The methods may comprise transacetylating the phosphonate herbicide. The phosphonate herbicides can comprise a CP bond and a CN bond and may be glyphosate.

REFERENCE TO PRIOR APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/213,791, filed Aug. 7, 2002, the disclosure of which is incorporatedherein by reference in its entirety; which is a divisional of U.S.application Ser. No. 09/441,340, filed Nov. 16, 1999, now U.S. Pat. No.6,448,476; which claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 60/108,763 filed Nov. 17, 1998.

FIELD OF THE INVENTION

The present invention relates in general to detoxifying phosphonateherbicides.

DESCRIPTION OF THE PRIOR ART

Phosphorous containing organic molecules can be naturally occurring orsynthetically derived. Organic molecules containing phosphorous-carbon(C—P) bonds are also found naturally or as synthetic compounds, and areoften not rapidly degraded, if at all, by natural enzymatic pathways.Synthetic organophosphonates and phosphinates, compounds that contain adirect carbon-phosphorous (C—P) bond in place of the better knowncarbon-oxygen-phosphorous linkage of phosphate esters (Metcalf et al.,Gene 129:27-32, 1993), have thus been widely used as insecticides,antibiotics, and as herbicides (Chen et al., J. Biol. Chem.265:4461-4471, 1990; Hilderbrand et al., The role of phosphonates inliving systems, Hilderbrand, R. L., ed, pp. 5-29, CRC Press, Inc., BocaRaton, Fla., 1983). Phosphonates are ubiquitous in nature, and are foundalone and in a diversity of macromolecular structures in a variety oforganisms (Jiang et al., J. Bacteriol. 177:6411-6421, 1995). Degradationof phosphonate molecules proceeds through a number of known routes, aC—P lyase pathway, a phosphonatase pathway, and a C—N hydrolysis pathway(Wanner, Biodegradation 5:175-184, 1994; Barry et al., U.S. Pat. No.5,463,175, 1995). Bacterial isolates capable of carrying out these stepshave been characterized (Shinabarger et al., J. Bacteriol. 168:702-707,1986; Kishore et al., J. Biol. Chem. 262:12, 164-12, 168, 1987; Pipke etal., Appl. Environ. Microbiol. 54:1293-1296, 1987; Jacob et al., Appl.Environ. Microbiol. 54:2953-2958, 1988; Lee et al., J. Bacteriol.174:2501-2510, 1992; Dumora et al., Biochim. Biophys. Acta 997:193-198,1989; Lacoste et al., J. Gen. Microbiol. 138:1283-1287, 1992). However,with the exception of phosphonatase and glyphosate oxidase (GOX), otherenzymes capable of carrying out these reactions have not beencharacterized.

Several studies have focused on the identification of genes required forC—P lyase degradation of phosphonates. Wackett et al. (J. Bacteriol.169:710-717, 1987) disclosed broad substrate specificity towardphosphonate degradation by Agrobacterium radiobacter and specificutilization of glyphosate as a sole phosphate source. Shinabarger et al.and Kishore et al. disclosed C—P lyase degradation of the phosphonateherbicide, glyphosate, to glycine and inorganic phosphate through asarcosine intermediate by Pseudomonas species.

E. coli B strains had previously been shown to be capable of phosphonateutilization (Chen et al.), whereas E. coli K-12 strains were incapableof phosphonate degradation. However, K-12 strains were subsequentlyshown to contain a complete, though cryptic, set of genes (psiD or phn)capable of phosphonate utilization (Makino et al.), as mutants wereeasily selected by growth on low phosphate media containing methyl- orethyl-phosphonate as sole phosphorous sources. Such K-12 strains adaptedfor growth on methyl- or ethylphosphonate were subsequently shown to beable to utilize other phosphonates as sole phosphorous sources (Wackettet al., J. Bacteriol. 169:1753-1756, 1987).

Avila et al. (J. Am. Chem. Soc. 109:6758-6764, 1987) were interested inthe mechanistic appraisal of biodegradative and detoxifying processes asrelated to aminomethyl-phosphonates, including elucidating theintermediates, products, and mechanisms of the degradativedephosphorylation process. Avila et al. studied the formation ofdephosphorylated biodegradation products from a variety ofaminophosphonate substrates in E. coli K-12 cultures previously adaptedto growth on ethylphosphonate. Furthermore, Avila et al. utilizedN-acetyl-AMPA (N-acetyl-amino-methyl-phosphonate) as a sole phosphatesource in some of their studies in order to show that acetylated AMPAwas not inhibitory to C—P bond cleavage. In addition, Avila et al. notedthat N-acetyl-AMPA was able to serve as a sole phosphate source duringE. coli K-12 growth, however, they did not observe N-acetyl-AMPAformation when AMPA was used as a sole phosphate source. Their resultsindicated that AMPA was not a substrate for acetylation in E. coli.

Chen et al. identified a functional psiD locus from E. coli B bycomplementation cloning into an E. coli K-12 strain deficient forphosphonate utilization, which enabled the K-12 strain to utilizephosphonate as a sole phosphate source (J. Biol. Chem. 265:4461-4471,1990). Chen et al. thus disclosed the DNA sequence of the psiDcomplementing locus, identified on a 15.5 kb BamHI fragment containing17 open reading frames designated phnA-phnQ, comprising the E. coli Bphn operon. The cryptic phn (psiD) operon from E. coli K-12 wassubsequently found to contain an 8-base pair insertion in phnE. Theresulting frameshift in phnE not only results in defective phnE geneproduct, but also apparently causes polar effects on the expression ofdownstream genes within the operon, which prevent phosphonateutilization (Makino et al., J. Bacteriol. 173:2665-2672, 1991). Theoperon has been more accurately described to contain the genes phnC-phnPby the work of Makino et al. Further research has been directed tounderstanding the nature of the function of each of the genes withinthis operon (Chen et al., J. Biol. Chem. 265:4461-4471, 1990; Makino etal., J. Bacteriol. 173:2665-2672, 1991; Wanner et al., FEMS Microbiol.Lett. 100:133-140, 1992; Metcalf et al., Gene 129:27-32, 1993; Ohtaki etal., Actinomyceteol. 8:66-68, 1994). In all of these efforts, the phnOgene has been implicated as a regulatory protein based on its similarityto other nucleotide binding proteins containing structuralhelix-turn-helix motifs. Furthermore, mutagenesis of genes in the phnoperon demonstrated that phnO was not required for phosphonateutilization, further supporting the proposed regulatory function forthis gene (Metcalf et al., J. Bacteriol. 173:587-600, 1991), at leastfor the phosphonates tested. Homologous phn sequences have beenidentified from other bacteria, including a gene substantially similarto E. coli phnO, isolated from S. griseus, using nucleotide sequencesdeduced from those in the E. coli phnO gene (Jiang et al., J. Bacteriol.177:6411-6421, (1995); McGrath et al., Eur. J. Biochem. 234:225-230,(1995); Ohtaki et al., Actinomyceteol. 8:66-68, (1994)). However, nofunction other than as a regulatory factor has been proposed for phnO. Aregulatory role for phnO in the CP lyase operon has been cited again ina recent review (Berlyn, Microbiol. Molec. Biol. Rev. 62:814-984, 1998).

Advances in molecular biology, and in particular in plant sciences incombination with recombinant DNA technology, have enabled theconstruction of recombinant plants which contain normative genes ofagronomic importance. Furthermore, when incorporated into and expressedin a plant, such genes desirably confer some beneficial trait orcharacteristic to the recombinant plant. One such trait is herbicideresistance. A recombinant plant capable of growth in the presence of aherbicide has a tremendous advantage over herbicide-susceptible species.In addition, herbicide tolerant plants provide a more cost effectivemeans for agronomic production by reducing the need for tillage tocontrol weeds and volunteers.

Chemical herbicides have been used for decades to inhibit plantmetabolism, particularly for agronomic purposes as a means forcontrolling weeds or volunteer plants in fields of crop plants. A classof herbicides which have proven to be particularly effective for thesepurposes are known as phosphonates or phosphonic acid herbicides.Perhaps the most agronomically successful phosphonate herbicide isglyphosate (N-phosphono-methyl-glycine).

Recombinant plants have been constructed which are tolerant to thephosphonate herbicide glyphosate. When applied to plants, glyphosate isabsorbed into the plant tissues and inhibits aromatic amino acidformation, mediated by an inhibition of the activity of theplastid-localized 5-enolpyruvyl-3-phosphoshikimic acid synthase enzyme,also known as EPSP synthase or EPSPS, an enzyme generally thought to beunique to plants, bacteria and fungi. Recombinant plants have beentransformed with a bacterial EPSPS enzyme which is much less sensitiveto glyphosate inhibition. Therefore, plants expressing this bacterialEPSPS are less sensitive to glyphosate, and are often characterized asbeing glyphosate tolerant. Therefore, greater amounts of glyphosate canbe applied to such recombinant plants, ensuring the demise of plantswhich are susceptible or sensitive to the herbicide. However, othergenes have been identified which, when transformed into a plant genome,encoding enzymes which also provide glyphosate tolerance. One suchenzyme has been described as GOX, or glyphosate-oxidoreductase. GOXfunctions in providing protection to plants from the phosphonateherbicide glyphosate by catalyzing the degradation of glyphosate toaminomethyl phosphonic acid (AMPA) and glyoxylate. AMPA produced as aresult of glyphosate degradation can cause bleaching and stunted ordepressed plant growth, among other undesirable characteristics. Manyplant species are also sensitive to exogeneously applied AMPA, as wellas to endogenous AMPA produced as a result of GOX mediated glyphosateherbicide degradation. No method has been described which discloses theprotection of plants from applications of phosphonate herbicides such asAMPA.

Barry et al. (U.S. Pat. No. 5,633,435) disclose genes encoding EPSPsynthase enzymes which are useful in producing transformed bacteria andplants which are tolerant to glyphosate as a herbicide, as well as theuse of such genes as a method for selectively controlling weeds in aplanted transgenic crop field. Barry et al. (U.S. Pat. No. 5,463,175)disclose genes encoding glyphosate oxidoreductase (GOX) enzymes usefulin producing transformed bacteria and plants which degrade glyphosateherbicide as well as crop plants which are tolerant to glyphosate as aherbicide. Barry et al. (U.S. Pat. No. 5,463,175) disclosed theformation of AMPA as a product of GOX mediated glyphosate metabolism.AMPA has been reported to be much less phytotoxic than glyphosate formost plant species (Franz, 1985) but not for all plant species (Maier,1983; Tanaka et al., 1986). Co-expression of a gene encoding a proteincapable of neutralizing or metabolizing AMPA produced by glyphosatedegradation would provide a substantial improvement over the use of GOXalone. Thus, a method for overcoming sensitivity to AMPA formation as aresult of glyphosate degradation, or a method for resistance to AMPAwhen used as a herbicide or as a selective agent in plant transformationmethods, would be useful for providing enhanced or improved herbicidetolerance in transgenic plants and in other organisms sensitive to suchcompounds.

The use of glyphosate as a chemical gametocide has been described (U.S.Pat. No. 4,735,649). Therein, it is disclosed that glyphosate can, underoptimal conditions, kill about 95% of male gametes, while leaving about40-60% of the female gametes capable of fertilization. In addition, astunting effect was typically observed at the application levelsdisclosed, shown by a reduction in the size of the plant and by a minoramount of chlorosis. Thus, a major drawback of using glyphosate as agametocide, as is generally true with most gametocides, is thephytotoxic side effects resulting from lack of sufficient selectivityfor male gametes. These phytotoxic manifestations may be effectuated byAMPA production in transgenic plants expressing GOX after treatment withglyphosate. Therefore, it would be advantageous to provide a method forpreventing the stunting effect and chlorosis as side effects of usingglyphosate as a gametocide in transgenic plants expressing GOX.Furthermore, a more effective method would optimally kill more than 95%of male gametes or prevent male gametes from maturing and would leavegreater than 60% of female gametes substantially unaffected. It isbelieved that tissue specific co-expression of GOX with a transacylasegene encoding an enzyme capable of N-acylation of AMPA would achievethis goal.

It has now been discovered that the E. coli phnO gene encodes an enzymehaving transacylase, acyltransferase, or Acyl-CoA transacylase activityin which a preferred substrate is a phosphonate displaying a terminalamine, and in particular amino-methyl-phosphonic acid (AMPA). Thetransfer of an acyl group from an Acyl-CoA to the free terminal amine ofAMPA results in the formation of an N-acylated AMPA. Plants are notknown to acylate AMPA to any great extent, and some plants have beenshown to be sensitive to AMPA and insensitive to acyl-AMPA. Thus,expression of phnO in plants would be useful in enhancing thephosphonate herbicide tolerance, particularly when AMPA is used as aherbicide or selective agent in plant transformation, and moreparticularly when glyphosate is used as a herbicide in combination withrecombinant plants expressing a GOX gene.

SUMMARY OF THE INVENTION

Briefly therefore the present invention is directed to a composition ofmatter comprising a novel class of genes which encode proteins capableof N-acylation of phosphonate compounds and to methods of using thesegenes and encoded proteins for improving plant tolerance to phosphonateherbicides. The present invention is also directed to a method forselecting recombinant plants and microbes transformed with genesencoding proteins which are capable of N-acylation of phosphonatecompounds, and to peptides which are capable of N-acylation of thecompound N-amino-methyl-phosphonic acid (N-AMPA) and other relatedphosphonate compounds. In addition, the present invention is alsodirected to a method for using plants transformed with transacylasegenes to prevent self-fertilization or to a method for enhancinghetero-fertilization in plants.

Among the several advantages found to be achieved by the presentinvention, therefore, may be noted the provision of producing stablytransformed herbicide tolerant recombinant plants which have insertedinto their genomes a polynucleotide sequence encoding a desired geneproduct, preferably an N-acyl-transferase enzyme. The polynucleotidesequence preferably is composed of a cassette containing a promotersequence which is functional in plants and which is operably linked 5′to a structural DNA sequence which, when transcribed into an RNAsequence, encodes an N-acyl-transferase enzyme peptide. The promotersequence can be heterologous with respect to the structural DNA sequenceand causes sufficient expression of the transferase enzyme in planttissue to provide herbicide tolerance to the plant transformed with thepolynucleotide sequence. The structural sequence is preferably operablylinked 3′ to a 3′ non-translated polyadenylation sequence whichfunctions in plants, and which when transcribed into RNA along with thestructural sequence causes the addition of a polyadenylated nucleotidesequence to the 3′ end of the transcribed RNA. Expression of thestructural DNA sequence produces sufficient levels of theacyltransferase enzyme in the plant tissue to enhance the herbicidetolerance of the transformed plant.

As a further embodiment, the structural DNA sequence may also contain anadditional 5′ sequence encoding an amino-terminal peptide sequence whichfunctions in plants to target the peptide produced from translation ofthe structural sequence to an intracellular organelle. This additionalcoding sequence is preferably linked in-frame to the structural sequenceencoding the acyltransferase enzyme. The amino terminal peptide sequencecan be either a signal peptide or a transit peptide. The intracellularorganelle can be a chloroplast, a mitochondrion, a vacuole, endoplasmicreticulum, or other such structure. The structural DNA sequence may alsobe linked to 5′ sequences such as untranslated leader sequences (UTL's),intron sequences, or combinations of these sequences and the like whichmay serve to enhance expression of the desired gene product. Intronsequences may also be introduced within the structural DNA sequenceencoding the acyltransferase enzyme. Alternatively, chloroplast orplastid transformation can result in localization of an acyltransferasecoding sequence and enzyme to the chloroplast or plastid, obviating therequirement for nuclear genome transformation, expression from thenuclear genome, and subsequent targeting of the gene product to asubcellular organelle.

Preferably, the recombinant plant expresses a gene encoding an enzymewhich catalyzes the formation of AMPA. AMPA formation can result fromthe metabolism of a naturally occurring precursor, from a precursor suchas glyphosate provided to the plant, or can result from the formation ofAMPA through some catabolic pathway. Co-expression of GOX along withAMPA acyltransferase expression provides a plant which is surprisinglymore resistant to certain phosphonate herbicides. However, oneembodiment allowing plants transformed with only an N-acyltransferase togrow in the presence of AMPA or similar or related compounds wouldprovide a useful selective method for identifying geneticallytransformed plants, callus, or embryogenic tissues.

In accordance with another aspect of the present invention is theprovision of a method for selectively enhancing or improving herbicidetolerance in a recombinant plant which has inserted into its nuclear,chloroplast, plastid or mitochondrial genome a cassette comprised of apolynucleotide sequence which encodes an N-acyl-transferase enzyme.

A further embodiment encompasses the improvement of a method forselectively enhancing herbicide tolerance in a transformed plantexpressing a GOX gene which encodes a glyphosate oxidoreductase enzymeexpressed in the same plants in which an acyltransferase enzyme isproduced.

In accordance with another aspect of the present invention is theprovision of a method for producing a genetically transformed herbicidetolerant plant by inserting into a genome of a plant cell a cassettecomprising a polynucleotide sequence which encodes an N-acyl-transferaseenzyme.

A further embodiment encompasses the improvement of a method forproducing a genetically transformed herbicide tolerant plant from aplant cell expressing a GOX gene which encodes a glyphosateoxidoreductase enzyme expressed in the same plant cell in which anacyltransferase enzyme is produced.

In any of the foregoing embodiments, the herbicide tolerant plant orplant cell can be selected from the group consisting of corn, wheat,cotton, rice, soybean, sugarbeet, canola, flax, barley, oilseed rape,sunflower, potato, tobacco, tomato, alfalfa, lettuce, apple, poplar,pine, eucalyptus, acacia, poplar, sweetgum, radiata pine, loblolly pine,spruce, teak, alfalfa, clovers and other forage crops, turf grasses,oilpalm, sugarcane, banana, coffee, tea, cacao, apples, walnuts,almonds, grapes, peanuts, pulses, petunia, marigolds, vinca, begonias,geraniums, pansy, impatiens, oats, sorghum, and millet.

In accordance with another aspect of the present invention is theprovision of a peptide capable of N-acylation of the compoundN-aminomethylphosphonic acid (N-AMPA or AMPA) or other such compoundswhich are capable of causing phytotoxic effects when applied to,introduced into, or produced by plant metabolisms. One such peptide isN-aminomethylphosphonic acid transacylase (AAT) derived from expressionof an E. coli phnO structural gene sequence. Other peptides similar instructure and function to the E. coli phnO gene product are alsocontemplated.

Another aspect of the present invention is the provision of a method forselecting cells transformed with a vector containing an acyltransferasegene expressing an enzyme capable of N-acylation of AMPA and likecompounds. The method includes the steps of transforming a population ofcells with the vector, and isolating and purifying the transformed cellsfrom non-transformed cells in the population after selecting for thetransformed cells by incubation in the presence of amounts of AMPAsufficient to be inhibitory to the growth or viability of anynon-transformed cells. The transformed cells can be bacterial, plant orfungal cells. Bacterial cells can be members of any of the familiesencompassed by Enterobacteraceae, Mycobacteraceae, Agrobacteraceae, andActinobacteraceae, among others. Fungal cells can be members ofAscomycota, Basidiomycota, etc. Plant cells can be derived from anymember of the Plantae family.

A further embodiment of the present invention provides for a method forproducing a plant from a tissue, a cell, or other part of a plant whichwas derived from a plant transformed with an acyltransferase gene, aphnO gene, a gox gene, a gene in which GOX and acyltransferase peptidesare produced from a translational fusion or a transcriptional fusion, ora polycistronic gene which encodes GOX and acyltransferase peptides.

A further embodiment of the present invention provides for a method forproducing plants which express all or a portion of a phnO gene orsimilar acyltransferase gene, or a GOX gene as an antisense gene in atissue specific manner.

Other aspects also include reagents such as antibodies directed to AMPAacyltransferase, and polynucleotides for use in identifyingacyltransferase gene sequences. These reagents can be included in kitscontaining AMPA acyltransferase, polynucleotides which are or arecomplimentary to an AMPA acyltransferase gene sequence, polynucleotidesfor use in thermal amplification of an AMPA acyltransferase genesequence, antibodies directed to AMPA acyltransferase for the detectionof AMPA acyltransferase in the laboratory or in the field, and any otherreagents necessary for use in kit form as well as for use in otherassays contemplated herein.

A further object of the present invention is to provide a method forusing phosphonate herbicides as chemical hybridizing agents. The methodallows for selective gametocidal effects and for the production of malesterile plants. Such plants may be engineered so that gox or phnO, orgox and phnO fail to be expressed in plant tissues required forreproduction, causing sensitivity to applied phytotoxic compounds whichinhibit formation of mature gamete structures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a [¹⁴C] isotope detection HPLC chromatogramrepresenting a sample of a dosing solution containing only [¹⁴C]glyphosate (11.3 minutes, 98.8%), and trace amounts of [¹⁴C] AMPA (5.8minutes, 0.16%) and an unidentified [¹⁴C] material (10.2 minutes, 1%).

FIG. 2 illustrates an HPLC profile of a mixture of standards of theobserved radioactive metabolites [¹⁴C] AMPA, [¹⁴C] glyphosate, andN-acetyl-[¹⁴C]-AMPA, as well as the impurity identified asN-acetyl-N-methyl-[¹⁴C]-AMPA.

FIG. 3 illustrates a representative HPLC profile of an extract from acorn callus tissue transformed with GOX and AMPA acetyltransferase, andtreated with [¹⁴C] glyphosate. The peaks indicate [¹⁴C] glyphosate (10.8minutes, 92.5% of total observed [¹⁴C]), [¹⁴C] AMPA primarily generatedby GOX mediated glyphosate degradation (5.98 minutes, 1.71% of totalobserved [¹⁴C]), and N-acetyl-[¹⁴C]AMPA produced from acylation of [¹⁴C]AMPA mediated by recombinant AMPA acyltransferase expressed withincallus tissue (13.29 minutes, 4.54% total observed [¹⁴C]).

FIG. 4 illustrates plasmid pMON17261.

FIG. 5 illustrates plasmid pMON32571.

FIG. 6 illustrates plasmid pMON32936.

FIG. 7 illustrates plasmid pMON32946.

FIG. 8 illustrates plasmid pMON32948.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the following detailed description should not be construed to undulylimit the present invention as modifications and variations in theembodiments discussed herein may be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

Many words and phrases are well known in the art of molecular biology,microbiology, protein chemistry, and plant sciences and generally havetheir plain and ordinarily understood meaning, otherwise to be taken incontext. However, the following words and phrases as used herein havethe meanings generally set forth below.

AMPA acyltransferase. As used herein, AMPA acyltransferase refers to anenzyme which functions in transferring an acyl chemical group from anacylcarrier compound such as coenzyme A, which is well known andabbreviated in the biological and chemical arts as CoA. In particular,an AMPA acyltransferase transfers an acyl chemical group from anacylcarrier to the free amino group of aminomethylphosphonate, wellknown to be a byproduct of glyphosate oxidoreductase mediated glyphosatemetabolism. AMPA acyltransferase (AAT), which herein may also be knownas AMPA acetyltransferase, AMPA transacylase, or acetyl-AMPA synthase(AAS), has been shown herein to be capable of acetyl transferaseactivity, propionyl transferase activity, malonyl transferase activity,and succinyl transferase activity. Thus, any biologically functionalequivalent of these compounds (acetyl, propionyl, malonyl, or succinyl)which serves as an acyl-carrier form of substrate capable of functioningwith an AMPA acyltransferase enzyme is within the scope of the presentinvention. One AMPA acyltranferase which has been identified, and shownby example herein to function according to the description containedherein, has previously been referred to in the art as PhnO, a proteinencoded by the phnO gene within the E. coli phn operon.

Biological functional equivalents. As used herein such equivalents withrespect to the AMPA-acyltransferase proteins of the present inventionare peptides, polypeptides and proteins that contain a sequence ormoiety exhibiting sequence similarity to the novel peptides of thepresent invention, such as PhnO, and which exhibit the same or similarfunctional properties as that of the polypeptides disclosed herein,including transacylase activity. Biological equivalents also includepeptides, polypeptides and proteins that react with, i.e. specificallybind to antibodies raised against PhnO and that exhibit the same orsimilar transacylase activity, including both monoclonal and polyclonalantibodies.

Biological functional equivalents as used herein with respect to genesencoding acyltransferases are polynucleotides which react with thepolynucleotide sequences contemplated and described herein, i.e. whichare capable of hybridizing to a polynucleotide sequence which is or iscomplementary to a polynucleotide encoding an acyltransferase whichfunctions in transacylation of AMPA or which encode substantiallysimilar acyltransferase proteins contemplated and described herein. Aprotein which is substantially similar to the proteins described hereinis a biological functional equivalent and exhibits the same or similarfunctional properties as that of the polypeptides disclosed herein,including improved herbicide tolerance or improved herbicide resistance.Biological equivalent peptides contain a sequence or moiety such as oneor more active sites which exhibit sequence similarity to the novelpeptides of the present invention, such as PhnO. Biological equivalentsalso include peptides, polypeptides, and proteins that react with, i.e.which specifically bind to antibodies raised against PhnO and PhnO-likepeptide sequences and which exhibit the same or similar improvement inherbicidal tolerance or resistance, including both monoclonal andpolyclonal antibodies.

Chloroplast or plastid localized, as used herein, refers to a biologicalmolecule, either polynucleotide or polypeptide, which is positionedwithin the chloroplast or plastid such that the molecule is isolatedfrom the cellular cytoplasmic milieu, and functions within thechloroplast or plastid cytoplasm to provide the effects claimed in theinstant invention. Localization of a biological molecule to thechloroplast or plastid can occur, with reference to polynucleotides, byartificial mechanical means such as electroporation, mechanicalmicroinjection, or by polynucleotide coated microprojectile bombardment,or with reference to polypeptides, by secretory or import means whereina natural, non-naturally occurring, or heterologous plastid orchloroplast targeting peptide sequence is used which functions totarget, insert, assist, or localize a linked polypeptide into achloroplast or plastid.

Event refers to a transgenic plant or plant tissue derived from theinsertion of foreign DNA into one or more unique sites in the nuclear,mitochondrial, plastid or chloroplast DNA.

Expression: The combination of intracellular processes, includingtranscription, translation, and other intracellular protein and RNAprocessing and stabilization functions, which a coding DNA molecule suchas a structural gene is subjected to in order to produce a gene product.

Non-naturally occurring gene: A non-naturally occurring acyl-transferasegene of the present invention contains genetic information encoding aplant functional RNA sequence, but preferably is a gene encoding anacyl-transferase protein, whether naturally occurring or a variant of anaturally occurring protein, prepared in a manner involving any sort ofgenetic isolation or manipulation. This includes isolation of the genefrom its naturally occurring state, manipulation of the gene as by codonmodification, site specific mutagenesis, truncation, introduction orremoval of restriction endonuclease cleavage sites, synthesis orresynthesis of a naturally occurring sequence encoding anacyltransferase of the present invention by in vitro methodologies suchas phosphoramidite chemical synthesis methods, etc., thermalamplification methods such as polymerase chain reaction, ligase chainreaction, inverted polymerase reaction, and the like etc., and any othermanipulative or isolative method.

Operably Linked: Nucleic acid segments connected in frame so that theproperties of one influence the expression of the other. For example, apromoter sequence having properties of polymerase loading, binding, andinitiation of transcription functions influences the expression ofsequences which are linked to the promoter.

Plant-Expressible Coding Regions: Coding regions which are expressible,i.e can be transcribed and/or translated in planta, because they containtypical plant regulatory elements to facilitate the expression of a geneof interest.

Plastid Transit Peptide: Any amino acid sequence useful in targeting orlocalizing a linked amino acid, such as a protein fusion, to asubcellular compartment or organelle such as a plastid or chloroplast.Amino acid sequences which facilitate entry into a mitochondria are notaltogether unlike or dissimilar from plastid transit peptides, and arealso described as transit peptides, but fail to function for targetingpeptide sequences to plastid or chloroplast organelles.

Progeny of a transgenic plant includes any offspring or descendant ofthe transgenic plant which contains at least one heterologous ortrans-gene, or any subsequent plant derived from the transgenic plantwhich has the transgene in its lineage. Progeny is not limited to onegeneration, but rather encompasses the descendants of the transgenicplant so long as they contain or express the desired transgene. Seedscontaining transgenic embryos as well as seeds from the transgenicplants and their offspring or descendants are also important parts ofthe invention. Transgenic cells, tissues, seeds or plants which containa desired transgene are progeny of the original transgenic cells,tissue, or plant.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provides an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

R₀ is the primary regenerant plant derived from transformation of planttissue or cells in culture. Subsequent progeny or generations derivedfrom the R₀ are referred to as R₁ (first generation), R₂ (secondgeneration), etc.

Regeneration: The process of producing a whole plant by growing a plantfrom a plant cell or plant tissue (e.g., plant protoplast or explant).

Structural Coding Sequence refers to a DNA sequence that encodes apeptide, polypeptide, or protein that is produced followingtranscription of the structural coding sequence to messenger RNA (mRNA),followed by translation of the mRNA to produce the desired peptide,polypeptide, or protein product.

Structural gene: A gene that is expressed to produce a polypeptide.

Substantial homology: As this term is used herein, substantial homologyrefers to nucleic acid sequences which are from about 40 to about 65percent homologous, from about 66 percent homologous to about 75 percenthomologous, from about 76 percent homologous to about 86 percenthomologous, from about 87 percent homologous to about 90 percenthomologous, from about 91 percent homologous to about 95 percenthomologous, and from about 96 percent homologous to about 99 percenthomologous to a reference polynucleotide sequence, such as either an E.coli phnO gene sequence. A first polynucleotide molecule which issubstantially homologous to a second polynucleotide molecule is or iscomplimentary to the second polynucleotide such that the firstpolynucleotide molecule hybridizes to the second polynucleotide moleculeor its complementary sequence under stringent hybridization conditions,with stringency being defined as the optimum concentration of salt andtemperature required to bring about hybridization of a firstpolynucleotide to a second polynucleotide. Methods for varyingstringency are well known in the art but may be referenced in Sambrooket al., Eds., Molecular Cloning: A Laboratory Manual, Second Edition,1989, Cold Spring Harbor Press; or Ausubel et al, Eds., Short Protocolsin Molecular Biology, Third Edition, 1995, John Wiley and Sons, Inc.Polypeptides which are believed to be within the scope if the presentinvention are those which are from about 40 to about 65 percent similar,from about 66 percent similar to about 75 percent similar, from about 76percent similar to about 86 percent similar, from about 87 percentsimilar to about 90 percent similar, from about 91 percent similar toabout 95 percent similar, and from about 96 percent similar to about 99percent similar to a reference polypeptide sequence, preferably to an E.coli PhnO peptide sequence.

Terminator: As used herein with respect to plant specific sequencesintended for in planta expression, the 3′ end transcription terminationand polyadenylation sequence.

Transformation is a process of introducing an exogenous polynucleotidesequence, such as a plasmid or viral vector or a recombinantpolynucleotide molecule, into a cell, protoplast, plastid orchloroplast, or mitochondria in which the exogenous polynucleotidesequence is either incorporated into an endogenous polynucleotidesequence contained within the cell, or is capable of autonomousreplication. A transformed cell is a cell which has been altered by theintroduction of one or more exogenous polynucleotide molecules into thatcell. A stably transformed cell is a transformed cell which hasincorporated all or a portion of the exogenous polynucleotide into thecells' nuclear, mitochondrial, or plastid or chloroplast genomicmaterial such that the exogenous polynucleotide confers some genotypicor phenotypic trait or traits to that cell and to the progeny of thetransformed cell, measured by the detection of the exogenouslyintroduced polynucleotide, the mRNA or protein product of the exogenouspolynucleotide, a metabolite not normally produced by or found withinthe cell in the absence of the exogenous polynucleotide, or a visualinspection of the cell, plant tissue, or plants derived from thetransformed cell.

Transgene: A transgene is a polynucleotide sequence which has beentransferred to a cell and comprises an expression cassette containing astructural gene sequence encoding a desired polypeptide. The transgeneis capable of being expressed when in a recipient transformed cell,tissue, or organism. This may include an entire plasmid or other vector,or may simply include the plant functional coding sequence of thetransferred polynucleotide. A transgenic cell is any cell derived fromor regenerated from a transformed cell, including the initiallytransformed cell. Exemplary transgenic cells include plant callus tissuederived from a transformed plant cell and particular cells such as leaf,root, stem, meristem, and other somatic tissue cells, or reproductive orgerm line and tapetal cells obtained from a stably transformedtransgenic plant. A transgenic event is a plant or progeny thereofderived from the insertion of at least one exogenous polynucleotide intothe nuclear, plastidic, or mitochondrial genome of a plant cell orprotoplast. A transgenic plant is a plant or a progeny thereof which hasbeen genetically modified to contain and express heterologouspolynucleotide sequences as proteins or as RNA or DNA molecules notpreviously a part of the plant composition. As specifically exemplifiedherein, a transgenic cotton plant, for example, is genetically modifiedto contain and express at least one heterologous DNA sequence operablylinked to and under the regulatory control of transcriptional andtranslational control sequences which function in plant cells or tissueor in whole plants. A transgenic plant may also be referred to as atransformed plant. A transgenic plant also refers to progeny of theinitial transgenic plant where those progeny contain and are capable ofexpressing the heterologous coding sequence under the regulatory controlof the plant expressible transcriptional and translational controlsequences described herein. A transgenic plant can produce transgenicflowers, seeds, bulbs, roots, tubers, fruit, and pollen and the like andcan be crossed by conventional breeding means with compatible lines ofplants to produce hybrid transgenic plants.

Vector: A DNA or other polynucleotide molecule capable of replication ina host cell and/or to which another DNA or other polynucleotide sequencecan be operatively linked so as to bring about replication of the linkedsequence. A plasmid is an exemplary vector.

In accordance with the present invention, it has been discovered thatplants can produce a phytotoxic compound when transformed with certaingenes encoding enzymes capable of degrading glyphosate. In particular,glyphosate oxidoreductase (GOX) mediated metabolism of glyphosateproduces a phytotoxic compound identified as N-aminomethyl-phosphonate(AMPA). Other studies have shown that an N-acylated derivative of AMPA,N-acyl-aminomethyl-phosphonate (N-acyl-AMPA or acyl-AMPA), is much lessphytotoxic to most plant species. Enzymes have been identified which areable to covalently modify AMPA through an acylation mechanism, resultingin the formation of N-acyl-AMPA. One enzyme in particular causesexogeneously applied AMPA to be N-acetylated. In plants expressing thisenzyme along with GOX, phytotoxic AMPA effects are not observed.

The inventions contemplated herein take advantage of recombinantpolynucleotide cassettes comprised of elements for regulating geneexpression into which sequences, such as structural genes encodinguseful proteins, can be inserted. Insertion of such sequences into anexpression cassette is preferably accomplished using restrictionendonucleases well known in the art, however other methods for insertionare known. For example, site specific recombination methods areeffective for inserting desired sequences into such expressioncassettes. Expression cassettes contain at least a plant operablepromoter for use in initiating the production of a messenger RNAmolecule from which the useful protein is translated. Cassettes alsocontain plant operable sequences, identified as 3′ sequences, whichfunction in terminating transcription and provide untranslated sequenceswhich are 3′ polyadenylated. Thus, an expression cassette intended foruse in plants should contain at least a promoter sequence linked at its3′ end to a 3′ transcription termination and polyadenylation sequence.Preferably, a polycloning sequence or linker sequence containing one ormore unique restriction endonuclease cleavage sites is present bridgingthe promoter and 3′ sequence for convenient insertion of structural genesequences and other elements. An expression cassette intended for use inplants also preferably contains a 5′ untranslated sequence insertedbetween the promoter and the 3′ sequence. 5′ untranslated sequences(UTL's) have been shown to enhance gene expression in plants. Intronsare also contemplated as sequences which may be present in suchexpression cassettes of the present invention. The presence of plantoperable introns has also been shown, in maize in particular, to enhancegene expression in certain plant species. Introns may be present in anexpression cassette in any number of positions along the sequence of thecassette. This can include positions between the promoter and the 3′termination sequence and/or within a structural gene. There may be morethan one intron present in an expression cassette, however for thepurposes of the contemplated inventions herein, it is preferred thatintrons be present when expression cassettes are used inmonocotyledonous plants and plant tissues. Enhancer sequences are alsowell known in the art and may be present, although not necessarily as apart of an expression cassette, as enhancer sequences are known tofunction when present upstream or downstream or even at great distancesfrom a promoter driving expression of a gene of interest.

The expression of a gene localized to the plant nuclear genome and whichexists in double-stranded DNA form involves transcription to produce aprimary messenger RNA transcript (mRNA) from one strand of the DNA byRNA polymerase enzyme, and the subsequent processing of the mRNA primarytranscript inside the nucleus. This processing involves a 3′non-translated polynucleotide sequence which adds polyadenylatenucleotides to the 3′ end of the RNA. Transcription of DNA into mRNA isregulated by a sequence of DNA usually referred to as the “promoter”.The promoter comprises a sequence of bases that signals RNA polymeraseto associate with the DNA and to initiate the transcription of mRNAusing the template DNA strand to make a corresponding complementarystrand of RNA.

Those skilled in the art will recognize that there are a number ofpromoters which are active in plant cells, and have been described inthe literature. Such promoters may be obtained from plants, plantviruses, or plant commensal, saprophytic, symbiotic, or pathogenicmicrobes and include, but are not limited to, the nopaline synthase(NOS) and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of Agrobacterium tumefaciens), the cauliflowermosaic virus (CaMV) 19S and 35S promoters, the light-inducible promoterfrom the small subunit of ribulose 1,5-bisphosphate carboxylase(ssRUBISCO, a very abundant plant polypeptide), the rice Act1 promoter,the Figwort Mosaic Virus (FMV) 35S promoter, the sugar cane bacilliformDNA virus promoter, the ubiquitin promoter, the peanut chlorotic streakvirus promoter, the comalina yellow virus promoter, the chlorophyll a/bbinding protein promoter, and meristem enhanced promoters Act2, Act8,Act11 and EF1a and the like. All of these promoters have been used tocreate various types of DNA constructs which have been expressed inplants (see e.g., McElroy et al., 1990; Barry and Kishore, U.S. Pat. No.5,463,175) and which are within the scope of the present invention.Chloroplast and plastid specific promoters, chloroplast or plastidfunctional promoters, and chloroplast or plastid operable promoters arealso envisioned. It is preferred that the particular promoter selectedshould be capable of causing sufficient in-planta expression to resultin the production of an effective amount of acyltransferase to render aplant substantially tolerant to phosphonate herbicides and products ofphosphonate herbicide metabolism. The amount of acyltransferase requiredto provide the desired tolerance may vary with the plant species.

One set of preferred promoters are constitutive promoters such as theCaMV35S or FMV35S promoters that yield high levels of expression in mostplant organs. Enhanced or duplicated versions of the CaMV35S and FMV35Spromoters are particularly useful in the practice of this invention (Kayet al, 1987; Rogers, U.S. Pat. No. 5,378,619). In addition, it may alsobe preferred to bring about expression of the acyltransferase gene inspecific tissues of the plant, such as leaf, stem, root, tuber, seed,fruit, etc., and the promoter chosen should have the desired tissue anddevelopmental specificity. Therefore, promoter function should beoptimized by selecting a promoter with the desired tissue expressioncapabilities and approximate promoter strength and selecting atransformant which produces the desired herbicide tolerance in thetarget tissues. This selection approach from the pool of transformantsis routinely employed in expression of heterologous structural genes inplants since there is variation between transformants containing thesame heterologous gene due to the site of gene insertion within theplant genome. (Commonly referred to as “position effect”). In additionto promoters which are known to cause transcription (constitutive ortissue-specific) of DNA in plant cells, other promoters may beidentified for use in the current invention by screening a plant cDNAlibrary for genes which are selectively or preferably expressed in thetarget tissues and then determine the promoter regions.

It is preferred that the promoters utilized have relatively highexpression in all meristematic tissues in addition to other tissuesinasmuch as it is now known that phosphonate herbicides can betranslocated and accumulated in this type of plant tissue.Alternatively, a combination of chimeric genes can be used tocumulatively result in the necessary overall expression level ofacyltransferase enzyme to result in the herbicide tolerant phenotype. Apromoter which provides relatively high levels of expression can causethe production of a desired protein to in planta levels ranging from 0.1milligrams per fresh weight gram of plant tissue, to 0.5 milligrams perfresh weight gram of plant tissue, to 1.0 milligrams per fresh weightgram of plant tissue, to 2.0 or more milligrams per fresh weight gram ofplant tissue. The in planta levels of a desired protein in geneticallyisogenic crops in a field can range across a spectrum, but generally thelevels fall within 70% of a mean, more preferably within 50% of a mean,and even more preferably within 25% of a mean for all plants analyzed ina given sample.

The promoters used in the DNA constructs (i.e. chimeric plant genes) ofthe present invention may be modified, if desired, to affect theircontrol characteristics. For example, the CaMV35S promoter may beligated to the portion of the Arabidopsis thalianaribulose-1,5-bisphosphate carboxylase small subunit gene (ssRUBISCO)that represses the expression of ssRUBISCO in the absence of light, tocreate a promoter which is active in leaves but not in roots. Theresulting chimeric promoter may be used as described herein. Forpurposes of this description, the phrase “CaMV35S” promoter thusincludes variations of CaMV35S promoter, e.g., promoters derived bymeans of ligation with operator regions, random or controlledmutagenesis, et cetera. Furthermore, the promoters may be altered tocontain multiple “enhancer sequences” to assist in elevating geneexpression. Examples of such enhancer sequences have been reported byKay et al. (1987).

One RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. Thenontranslated or 5′ untranslated leader sequence (NTR or UTR) can bederived from an unrelated promoter or coding sequence. For example, the5′ non-translated regions can also be obtained from viral RNA's, fromsuitable eucaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs, as presented in one ofthe following examples, wherein the non-translated region is derivedfrom the 5′ non-translated sequence that accompanies the promotersequence. Examples of plant gene leader sequences which are useful inthe present invention are the wheat chlorophyll a/b binding protein(cab) leader and the petunia heat shock protein 70 (hsp70) leader(Winter et al., 1988).

For optimal expression in monocotyledonous plants, an intron should alsobe included in the DNA expression construct. This intron would typicallybe placed near the 5′ end of the mRNA in untranslated sequence. Thisintron could be obtained from, but not limited to, a set of intronsconsisting of the maize hsp70 intron (Brown et al., U.S. Pat. No.5,424,412; 1995) or the rice Act1 intron (McElroy et al., 1990).

Where more than one expression cassette in included within a plasmid orother polynucleotide construct, a first expression cassette comprising aDNA molecule typically contains a constitutive promoter, a structuralDNA sequence encoding a glyphosate oxidoreductase enzyme (GOX), and a 3′non-translated region. A second expression cassette comprising a DNAmolecule typically contains a constitutive promoter, a structural DNAsequence encoding an N-acyl-transferase enzyme which is capable ofreacting with AMPA to produce N-acyl-AMPA, and a 3′ non-translatedregion. Additional expression cassettes comprising a DNA molecule arealso envisioned. For example, genes encoding insecticidal or fungicidalactivities, drought or heat tolerance, antibiotic compounds,pharmaceutical compounds or reagents such as tumor suppressor proteinsor antibody components, biopolymers, other commercially useful compoundsand the like may also be expressed in the plants envisioned by thepresent invention, along with genes which provide increased herbicidetolerance. A number of constitutive promoters which are active in plantcells have been described. Suitable promoters for constitutiveexpression of either GOX or an N-acyl-transferase include, but are notlimited to, the cauliflower mosaic virus (CaMV) 35S promoter (Odell etal. 1985), the Figwort mosaic virus (FMV) 35S (Sanger et al. 1990), thesugarcane bacilliform DNA virus promoter (Bouhida et al., 1993), thecommelina yellow mottle virus promoter (Medberry and Olszewski 1993),the light-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al.,1984), the rice cytosolic triosephosphate isomerase (TPI) promoter (Xuet al. 1994), the adenine phosphoribosyltransferase (APRT) promoter ofArabidopsis (Moffatt et al. 1994), the rice actin 1 gene promoter (Zhonget al. 1996), and the mannopine synthase and octopine synthase promoters(Ni et al. 1995). All of these promoters have been used to createvarious types of plant-expressible recombinant DNA constructs.Comparative analysis of constitutive promoters by the expression ofreporter genes such as the uidA (β-glucuronidase) gene from E. coli hasbeen performed with many of these and other promoters (Li et al. 1997;Wen et al. 1993).

Promoters used in the second cassette comprising a DNA molecule can beselected to control or limit specific expression where cell lethality isdesired. In a preferred embodiment, the promoter will be capable ofdirecting expression exclusively or primarily in tissues critical forplant survival or plant viability, while limiting expression of thesecond cassette comprising a DNA molecule in other nonessential tissues.For example, tissues which differentiate into pollen development orterminal tissues such as the pollen itself, the tapetal cell layer ofthe anther, or the anther tissues. Alternatively, plant promoterscapable of regulating the expression of genes in particular cell andtissue types are well known. Those that are most preferred in theembodiments of this invention are promoters which express specificallyduring the development of the male reproductive tissue or in pollen atlevels sufficient to produce inhibitory RNA molecules complementary tothe sense RNA transcribed by the constitutive promoter of the firstexpression cassette comprising a DNA molecule. Examples of these typesof promoters include the TA29 tobacco tapetum-specific promoter (Marianiet al. 1990), the PA1 and PA2 chalcone flavonone isomerase promotersfrom petunia (van Tunen et al. 1990), the SLG gene promoter fromBrassica oleracea (Heizmann et al. 1991), and LAT gene promoters fromtomato (Twell et al. 1991).

Anther and pollen-specific promoters from rice have been isolated.Examples include the Osg6B promoter, which was shown to drive expressionof the β-glucuronidase gene in transgenic rice in immature anthers. Noactivity was detected in other tissues of spikelets, leaves or roots(Yokoi et al. 1997). The PS1 pollen-specific promoter from rice has beenshown to specifically express the β-glucuronidase gene in rice pollen(Zou et al. 1994). Additional rice genes have been identified thatspecifically express in the anther tapetum of rice (Tsuchiya et al.1994, Tsuchiya et al. 1997). The isolation of additional genes expressedpredominantly during anther development in rice can be performed, forexample, by construction of a cDNA library to identify anther specificclones (Qu et al.).

Those skilled in the art are aware of the approaches used in theisolation of promoters which function in plants, and from genes ormembers of gene families that are highly expressed in particular planttissues such as in roots, shoots, meristem, leaves, flowers, fruits, inpollen, or in plant cell types involved in the production of pollen(Stinson et al. 1987; Brown and Crouch. 1990; McCormick et al. 1989).Further examples of tissue specific promoters include the promoter forthe exopolygalacturonase gene of maize (Dubald, et al. 1993) and thepromoter for the Zmc13 mRNA (Hanson, et al. 1989). Promoters which havebeen shown to preferentially express in tomato pollen are the LAT52 andLAT59 promoters (Twell et al. 1991). A portion of the maize pZtappromoter sequence (psgB6-1) was disclosed in U.S. Pat. No. 5,470,359.

A recombinant DNA molecule of the present invention typically comprisesa promoter operably or operatively linked to a DNA sequence encoding a5′ non-translated region, a DNA sequence of a plant intron, a structuralsequence encoding a chloroplast transit peptide (CTP), a DNA codingsequence for a gene encoding improved herbicide tolerance, and a 3′non-translated region.

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous DNA sequence, and can bespecifically modified if desired so as to increase translation of mRNA.A 5′ non-translated region can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencewhich accompanies the promoter sequence. The leader sequence could alsobe derived from an unrelated promoter or coding sequence.

The 3′ non-translated region of a plant operable recombinant DNAmolecule contains a polyadenylation signal which functions in plants tocause the addition of adenylate nucleotides to the 3′ end of the RNA.The 3′ non-translated region can be obtained from various genes whichare expressed in plant cells. The nopaline synthase 3′ untranslatedregion (Fraley et al. 1983), the 3′ untranslated region from peassRUBISCO (Coruzzi et al. 1994), the 3′ untranslated region from soybean7S seed storage protein gene (Schuler et al. 1982) and the pea smallsubunit of the pea ssRUBISCO gene are commonly used in this capacity.The 3′ transcribed, non-translated regions containing the polyadenylatesignal of Agrobacterium tumor-inducing (Ti) plasmid genes are alsosuitable.

Examples of plant introns suitable for expression in monocots includes,for example, maize hsp70 intron, rice actin 1 intron, maize ADH 1intron, Arabidopsis SSU intron, Arabidopsis EPSPS intron, petunia EPSPSintron and others known to those skilled in the art.

It may be particularly advantageous to direct the localization ofproteins conferring herbicide tolerance to subcellular compartment, forexample, to the mitochondrion, endoplasmic reticulum, vacuoles,chloroplast or other plastidic compartment. Proteins can be directed tothe chloroplast by including at their amino-terminus a chloroplasttransit peptide (CTP). Naturally occurring chloroplast targetedproteins, synthesized as larger precursor proteins containing anamino-terminal chloroplast targeting peptide directing the precursor tothe chloroplast import machinery, have been previously identified andare well known in the art. Chloroplast targeting peptides are generallycleaved by specific endoproteases located within the chloroplastorganelle, thus releasing the targeted mature and preferably activeenzyme from the precursor into the chloroplast melieu. Examples ofsequences encoding peptides which are suitable for directing thetargeting of the herbicide tolerance gene or transacylase gene productto the chloroplast or plastid of the plant cell include the petuniaEPSPS CTP, the Arabidopsis EPSPS CTP2 and intron, and others known tothose skilled in the art. Such targeting sequences provide for thedesired expressed protein to be transferred to the cell structure inwhich it most effectively functions, or by transferring the desiredexpressed protein to areas of the cell in which cellular processesnecessary for desired phenotypic function are concentrated. Chloroplasttargeting peptides have been found to be particularly useful in theselection of glyphosate resistant plants (Barry et al., U.S. Pat. No.5,463,175; Barry et al., U.S. Pat. No. 5,633,435). Glyphosate functionsto kill the cell by inhibiting aromatic amino acid biosynthesis whichtakes place within the chloroplast. Therefor, concentrating theresistance gene product within the chloroplast provides increasedresistance to the herbicide. The examples herein provide for atransacylase which is also targeted to or localized to and concentratedwithin the chloroplast. Specific examples of chloroplast targetingpeptides are well known in the art and include the Arabidopsis thalianaribulose bisphosphate carboxylase small subunit ats1A transit peptide,an Arabidopsis thaliana EPSPS transit peptide and a Zea maize ribulosebisphosphate carboxylase small subunit transit peptide. One CTP that hasfunctioned herein to localize heterologous proteins to the chloroplastwas derived from the Arabidopsis thaliana ribulose bisphosphatecarboxylase small subunit ats1A transit peptide. A polynucleotidesequence encoding a variant of this transit peptide used herein providesthe native transit peptide amino acid sequence plus a reiteration of thetransit peptide cleavage site, and has been shown herein to be usefulfor deploying active recombinant transacylase enzyme to the chloroplast.(SEQ ID NO:9).

An alternative means for localizing plant operable herbicide toleranceor herbicide resistance genes to a chloroplast or plastid includeschloroplast or plastid transformation. Recombinant plants can beproduced in which only the mitochondrial or chloroplast DNA has beenaltered to incorporate the molecules envisioned in this application.Promoters which function in chloroplasts have been known in the art(Hanley-Bowden et al., Trends in Biochemical Sciences 12:67-70, 1987).Methods and compositions for obtaining cells containing chloroplastsinto which heterologous DNA has been inserted have been described, forexample by Daniell et al. (U.S. Pat. No. 5,693,507; 1997) and Maliga etal. (U.S. Pat. No. 5,451,513; 1995).

The accumulation of AMPA in plants can cause phytotoxic symptoms whichare manifested phenotypically as chlorosis of the leaves, stuntedgrowth, infertility, and death, although not all of these symptoms areevidenced in every species of plant. It has been discovered herein thatenzymatic modification of the AMPA molecule by transacylation to produceN-acyl-AMPA provides a means for overcoming the phytotoxic effects ofAMPA. A method for assaying the conversion of AMPA to N-acyl-AMPAinvolves providing [¹⁴C] labeled AMPA as one substrate for thetransacylase enzyme, and acyl-CoA as another substrate for the enzyme inan aqueous reaction volume, and separating the [¹⁴C] labeled AMPAsubstrate from N-acyl-[¹⁴C]-AMPA product by HPLC on an anion exchangecolumn as described in the examples herein. Surprisingly, thetransacylase enzyme has been shown to be capable of utilizing otheracylated-CoA compounds as substrates for transacylating the AMPAsubstrate. In particular, propionyl-CoA was shown to be a particularlyreactive substrate for the transacylation reaction in vitro, producingN-propionyl-[¹⁴C]-AMPA. Larger acylated-CoA compounds, i.e. butyryl-CoAor methylmalonyl-CoA and other organic molecules covalently linked toCoA which have a carbon chain length greater than C₃ proved to be lesseffective in the transacylation reaction when using AMPA as theacyl-group recipient substrate. Notwithstanding this information, oneskilled in the art would recognize that other transacylases which aresubstantially related by amino acid sequence homology to a PhnO orPhnO-like enzyme as characterized herein would have a similar substratespecificity in the AMPA transacylase reaction as compared to thatencompassed by PhnO. These other enzymes too are conceptually within thescope and spirit of the invention described herein. For example, fattyacid biosynthesis is mediated by a wide range of acyl-CoA andacyl-carrier protein compounds which may be useful as substrates intransacylating phytotoxic compounds such as AMPA. A transacylase capableof AMPA transacylation using a fatty acid intermediate could conceivablyprovide plant protection by eliminating AMPA phytotoxicity. An enzymesuch as PhnO, which is capable of transacylation, may be useful indetoxifying a wide range of toxic compounds which contain CP bonds andwhich additionally contain a CN linkage.

Methods and compositions for transforming a bacterium, a yeast or fungalcell, a plant cell, or an entire plant with one or more expressionvectors comprising a phnO- or phnO-like gene sequence are furtheraspects of this disclosure. A transgenic bacterium, yeast or fungalcell, plant cell, or plant derived from such a transformation process orthe progeny and seeds from such a transgenic plant are also furtherembodiments of this invention.

Methods for transforming bacteria and yeast or fungal cells are wellknown in the art. Typically, means of transformation are similar tothose well known means used to transform other bacteria, such as E.coli, or yeast, such as Saccharomyces cerevisiae. Methods for DNAtransformation of plant cells include, but are not limited toAgrobacterium-mediated plant transformation, protoplast transformation,gene transfer into pollen, injection into reproductive organs, injectioninto immature embryos, plastid or chloroplast transformation, andparticle bombardment. Each of these methods has distinct advantages anddisadvantages. Thus, one particular method of introducing genes into aparticular plant species may not be the most effective for another plantspecies, but it is well known by those skilled in the art which methodsare useful for a particular plant species.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,binary bacterial artificial chromosome (BIBAC) vectors (Hamilton et al.,1996), direct delivery of DNA such as, for example by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973;Zatloukal et al., 1992); (2) physical methods such as microinjection(Capecchi, 1980), electroporation (Wong and Neuman, 1982; Fromm et al.,1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston and Tang,1994; Fynan et al., 1993; Luthra et al., 1997); (3) viral vectors(Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a; 1988b); and(4) receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner etal., 1992)

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135; U.S. Pat. No.5,518,908), soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011;McCabe et al. (1988); Christou et al. (1988)), Brassica (U.S. Pat. No.5,463,174), peanut (Cheng et al. (1996); De Kathen and Jabobsen (1990)).

Transformation of monocots using electroporation, particle bombardment,and Agrobacterium have also been reported. Transformation and plantregeneration have been achieved in asparagus (Bytebier et al. (1987)),barley (Wan and Lemaux (1994)), maize (Rhodes et al. (1988); Ishida etal. (1996); Gordon-Kamm et al. (1990); Fromm et al. (1990); Koziel etal. (1993); Armstrong et al. (1995), oat (Somers et al. (1992)),orchardgrass (Horn et al. (1988)), rice (Toriyama et al. (1988); Park etal. (1996); Abedinia et al. (1997); Zhang and Wu (1988); Zhang et al.(1988); Battraw and Hall (1990); Christou et al. (1991); Park et al.(1996)), rye (De la Pena et al. (1987)), sugar cane (Bower and Birch(1992)), tall fescue (Wang et al. (1992)), and wheat (Vasil et al.(1992); Weeks et al. (1993)). Techniques for monocot transformation andplant regeneration are also discussed in Davey et al. (1986).

Recombinant plants could also be produced in which only themitochondrial or chloroplast DNA has been altered to incorporate themolecules envisioned in this application. Promoters which function inchloroplasts have been known in the art (Handley-Bowden et al., Trendsin Biochemical Sciences 12:67-70, 1987). Methods and compositions forobtaining cells containing chloroplasts into which heterologous DNA hasbeen inserted has been described by Daniell et al., U.S. Pat. No.5,693,507 (1997) and Maliga et al. (U.S. Pat. No. 5,451,513; 1995).Recombinant plants which have been transformed using heterologous DNA,altering both nuclear and chloroplast or plastidic genomes is alsowithin the scope of this invention.

The present invention discloses DNA constructs comprising polynucleotidesequences encoding AMPA-transacylase. Methods for identifying andisolating heterologous genes encoding peptides which function inN-acylation of AMPA are disclosed herein. Methods for the constructionand expression of synthetic genes in plants are well known by those ofskill in the art and are described in detail in U.S. Pat. No. 5,500,365,and in monocotyledonous plants in particular in U.S. Pat. No. 5,689,052.The present invention contemplates the use of AMPA acyltransferase genesalone or in combination with genes encoding GOX mediated glyphosatedegradation enzymes in the transformation of both monocotyledonous anddicotyledonous plants. To potentiate the expression of these genes, thepresent invention provides DNA constructs comprising polynucleotidesequences encoding these types of proteins which are localized to theplant cell cytoplasm as well as sequences encoding plastid targetingpeptides positioned upstream of the polynucleotide sequences encodingthe AMPA transacylase and/or GOX proteins.

In one aspect, nucleotide sequence information provided by the inventionallows for the preparation of relatively short DNA sequences having theability to specifically hybridize to gene sequences of the selectedpolynucleotides disclosed herein. In these aspects, nucleic acid probesof an appropriate length are prepared based on a consideration ofselected polynucleotide sequences encoding AMPA transacylasepolypeptides, e.g., sequences such as are shown in SEQ ID NO:1, SEQ IDNO:2, and SEQ ID NO:3. Such nucleic acid probes may also be preparedbased on a consideration of selected polynucleotide sequences encoding aplastid targeting peptide, such as those shown in SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, and SEQ ID NO:14. The ability of such nucleic acidprobes to specifically hybridize to a gene sequence encoding an AMPAtransacylase polypeptide or a plastid targeting peptide sequence lendsto them particular utility in a variety of embodiments. Mostimportantly, the probes may be used in a variety of assays for detectingthe presence of complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined sequence of a AMPA transacylase gene from any suitable organismusing PCR™ technology. The process may also be used to detect, amplifyor mutate a defined sequence of the polynucleotide encoding a plastidtargeting peptide. Segments of genes related to the polynucleotidesencoding the AMPA transacylase polypeptides and plastid targetingpeptides of the present invention may also be amplified by PCR™ usingsuch primers.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes sequences that are substantiallycomplementary to at least a length of 14 to 30 or so consecutivenucleotides of a polynucleotide sequence flanking, in cis with, orencoding an AMPA transacylase, such as that shown in SEQ ID NO:5 or SEQID NO:6, or sequences that are substantially complementary to at least alength of 14 to 30 or so consecutive nucleotides of a sequence encodinga plastid targeting peptide. By “substantially complimentary”, it ismeant that a polynucleotide is preferably about 70% complimentary, ormore preferably about 80% complimentary, or even more preferably about90% complimentary, or most preferably about 99-100% complimentary insequence to a target polynucleotide sequence.

A size of at least 14 nucleotides in length helps to ensure that thefragment will be of sufficient length to form a duplex molecule that isboth stable and selective. Molecules having complementary sequences oversegments greater than 14 bases in length are generally preferred. Inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained,one will generally prefer to design nucleic acid molecules havinggene-complementary sequences of 14 to 20 nucleotides, or even longerwhere desired. Such fragments may be readily prepared by, for example,directly synthesizing the fragment by chemical means, such asphosphoramidite chemistries for example; by application of nucleic acidreproduction technology, such as the PCR™ technology of U.S. Pat. Nos.4,683,195, and 4,683,202 (each specifically incorporated herein byreference); or by excising selected DNA fragments from recombinantplasmids containing appropriate inserts and suitable restriction sites.

The present invention also contemplates an expression vector comprisinga polynucleotide of the present invention. Thus, in one embodiment anexpression vector is an isolated and purified DNA molecule comprising apromoter operably linked to a coding region that encodes a polypeptideof the present invention, which coding region is operatively linked to atranscription-terminating region, whereby the promoter drives thetranscription of the coding region. The coding region may include asegment or sequence encoding a AMPA transacylase and a segment orsequence encoding a plastid targeting peptide. The DNA moleculecomprising the expression vector may also contain a plant functionalintron, and may also contain other plant functional elements such assequences encoding untranslated sequences (UTL's) and sequences whichact as enhancers of transcription or translation.

As used herein, the terms “operatively linked” or “operably linked” meanthat a sequence which functions as a promoter is connected or linked toa coding region in such a way that the transcription of that codingregion is controlled and regulated by that promoter. Means foroperatively linking a promoter to a coding region to regulate bothupstream and downstream are well known in the art.

Preferred plant transformation vectors include those derived from a Tiplasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g.,by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and Eur. Pat.Appl. No. EP 0120516 (each specifically incorporated herein byreference). In addition, plant preferred transformation vectors directedto chloroplast or plastid transformation include those disclosed in U.S.Pat. No. 5,693,507 (1997), U.S. Pat. No. 5,451,513 (1995), McBride etal. (1995), Staub et al. (1995a), Staub et al. (1995b), and WO 95/24492(each specifically incorporated herein by reference).

Where an expression vector of the present invention is to be used totransform a plant, a promoter is selected that has the ability to driveexpression in that particular species of plant. Promoters that functionin different plant species are also well known in the art. Promotersuseful in expressing the polypeptide in plants are those which areinducible, viral, synthetic, or constitutive as described (Odell et al.,1985), and/or temporally regulated, spatially regulated, andspatio-temporally regulated. Preferred promoters include the enhancedCaMV35S promoters, and the FMV35S promoter.

The expression of a gene which exists in double-stranded DNA formlocalized to the plant nuclear genome involves transcription ofmessenger RNA (mRNA) from the coding strand of the DNA by an RNApolymerase enzyme, and the subsequent processing of the mRNA primarytranscript inside the nucleus. Genes expressed from within a chloroplastor plastid also produce an mRNA transcript which is not processedfurther prior to translation. In any event, transcription of DNA intomRNA is regulated by a region of DNA referred to as the “promoter”. TheDNA comprising the promoter is represented by a sequence of bases thatsignals RNA polymerase to associate with the DNA and to initiate thetranscription of mRNA using one of the DNA strands as a template to makea corresponding strand of RNA. The particular promoter selected shouldbe capable of causing sufficient expression of an AMPA acyltransferaseenzyme coding sequence to result in the production of an herbicidetolerance effective or herbicide resistance effective amount of thetransacylase protein localized to the desired intracellular location.

Structural genes can be driven by a variety of promoters in planttissues. Promoters can be near-constitutive (i.e. they drivetranscription of the transgene in all tissue), such as the CaMV35Spromoter, or tissue-specific or developmentally specific promotersaffecting dicots or monocots. Where the promoter is a near-constitutivepromoter such as CaMV35S or FMV35S, increases in polypeptide expressionare found in a variety of transformed plant tissues and most plantorgans (e.g., callus, leaf, seed, stem, meristem, flower, and root).Enhanced or duplicate versions of the CaMV35S and FMV35S promoters areparticularly useful in the practice of this invention (Kay et al., 1987;Rogers, U.S. Pat. No. 5,378,619).

Those skilled in the art will recognize that there are a number ofpromoters which are active in plant cells, and have been described inthe literature. Such promoters may be obtained from plants or plantviruses and include, but are not limited to, the nopaline synthase (NOS)and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of A. tumefaciens), the cauliflower mosaic virus(CaMV) 19S and 35S promoters, the light-inducible promoter from thesmall subunit of ribulose 1,5-bisphosphate carboxylase (ssRUBISCO, avery abundant plant polypeptide), the rice Act1 promoter and the FigwortMosaic Virus (FMV) 35S promoter. All of these promoters have been usedto create various types of DNA constructs which have been expressed inplants (see e.g., McElroy et al., 1990, U.S. Pat. No. 5,463,175).

In addition, it may also be preferred to bring about expression of genessuch as an AMPA acyltransferase which improve herbicide tolerance orherbicide resistance in specific tissues of a plant by using plantintegrating vectors containing a tissue-specific promoter. Specifictarget tissues may include the leaf, stem, root, tuber, seed, fruit,etc., and the promoter chosen should have the desired tissue anddevelopmental specificity. Therefore, promoter function should beoptimized by selecting a promoter with the desired tissue expressioncapabilities and approximate promoter strength, and selecting atransformant which produces the desired transacylase activity in thetarget tissues. This selection approach from the pool of transformantsis routinely employed in expression of heterologous structural genes inplants since there is variation between transformants containing thesame heterologous gene due to the site of gene insertion within theplant genome (commonly referred to as “position effect”). In addition topromoters which are known to cause transcription (constitutive ortissue-specific) of DNA in plant cells, other promoters may beidentified for use in the current invention by screening a plant cDNAlibrary for genes which are selectively or preferably expressed in thetarget tissues, then determining the promoter regions. Chloroplast orplastid functional promoters are known in the art (Hanley-Bowden et al.,Daniell et al., Maliga et al.).

Other exemplary tissue-specific promoters are corn sucrose synthetase 1(Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989),corn light harvesting complex (Simpson, 1986), corn heat shock protein(Odell et al., 1985), pea small subunit RuBP carboxylase (Poulsen etal., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase(McBride and Summerfelt, 1989), Ti plasmid nopaline synthase (Langridgeet al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), beanglycine rich protein 1 (Keller et al., 1989), CaMV 35S transcript (Odellet al., 1985) and Potato patatin (Wenzler et al., 1989) promoters.Preferred promoters are the cauliflower mosaic virus (CaMV 35S) promoterand the S-E9 small subunit RuBP carboxylase promoter.

The promoters used in the DNA constructs of the present invention may bemodified, if desired, to affect their control characteristics. Forexample, the CaMV35S promoter may be ligated to the portion of thessRUBISCO gene that represses the expression of ssRUBISCO in the absenceof light, to create a promoter which is active in leaves but not inroots. For purposes of this description, the phrase “CaMV35S” promoterthus includes variations of CaMV35S promoter, e.g., promoters derived bymeans of ligation with operator regions, random or controlledmutagenesis, etc. Furthermore, the promoters may be altered to containmultiple “enhancer sequences” to assist in elevating gene expression.Examples of such enhancer sequences have been reported by Kay et al.(1987). Chloroplast or plastid specific promoters are known in the art(Daniell et al., U.S. Pat. No. 5,693,507; herein incorporated byreference). Promoters obtainable from chloroplast genes, for example,such as the psbA gene from spinach or pea, the rbcL and atpB promoterregions from maize, and rRNA promoters. Any chloroplast or plastidoperable promoter is within the scope of the present invention.

A transgenic plant of the present invention produced from a plant celltransformed with a tissue specific promoter can be crossed with a secondtransgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

The RNA produced by a DNA construct of the present invention may alsocontain a 5′ non-translated leader sequence (5′UTL). This sequence canbe derived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence. One plant gene leader sequencefor use in the present invention is the petunia heat shock protein 70(hsp70) leader (Winter et al., 1988).

5′ UTL's are capable of regulating gene expression when localized to theDNA sequence between the transcription initiation site and the start ofthe coding sequence. Compilations of leader sequences have been made topredict optimum or sub-optimum sequences and generate “consensus” andpreferred leader sequences (Joshi, 1987). Preferred leader sequences arecontemplated to include those which comprise sequences predicted todirect optimum expression of the linked structural gene, i.e. to includea preferred consensus leader sequence which may increase or maintainmRNA stability and prevent inappropriate initiation of translation. Thechoice of such sequences will be known to those of skill in the art inlight of the present disclosure. Sequences that are derived from genesthat are highly expressed in plants, and in maize in particular, will bemost preferred. One particularly useful leader may be the petunia HSP70leader.

In accordance with the present invention, expression vectors designed tospecifically potentiate the expression of the polypeptide in thetransformed plant may include certain regions encoding plastid orchloroplast targeting peptides, herein abbreviated in various forms asCTP, CTP1, CTP2, etc., each representing a different or varianttargeting peptide sequence. These regions allow for the cellularprocesses involved in transcription, translation and expression of theencoded protein to be fully exploited when associated with certain GOXor AMPA transacylase protein sequences. Such targeting peptides functionin a variety of ways, such as for example, by transferring the expressedprotein to the cell structure in which it most effectively operates, orby transferring the expressed protein to areas of the cell in whichcellular processes necessary for expression are concentrated. The use ofCTP's may also increase the frequency of recovery of morphologicallynormal plants, and the frequency at which transgenic plants may berecovered.

Chloroplast targeting peptides have been found particularly useful inthe glyphosate resistant selectable marker system. In this system,plants transformed to express a protein conferring glyphosate resistanceare transformed along with a CTP that targets the peptide to the plantcell's chloroplasts. Glyphosate inhibits the shikimic acid pathway whichleads to the biosynthesis of aromatic compounds including amino acidsand vitamins. Specifically, glyphosate inhibits the conversion ofphosphoenolpyruvic acid and 3-phosphoshikimic acid to5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase or EPSPS).Introduction of a transgene encoding EPSPS allows the plant cell toresist the effects of glyphosate, especially when the transgene encodesa glyphosate insensitive EPSPS enzyme. Thus, as the herbicide glyphosatefunctions to kill the cell by interrupting aromatic amino acidbiosynthesis, particularly in the cell's chloroplast, the CTP allowsincreased resistance to the herbicide by concentrating what glyphosateresistance enzyme the cell expresses in the chloroplast, i.e. in thetarget organelle of the cell. Exemplary herbicide resistance enzymesinclude EPSPS and glyphosate oxido-reductase (GOX) genes (see Comai,1985, U.S. Pat. No. 4,535,060, specifically incorporated herein byreference in its entirety).

CTPs can target proteins to chloroplasts and other plastids. Forexample, the target organelle may be the amyloplast. Preferred CTP's ofthe present invention include those targeting both chloroplasts as wellas other plastids. Specific examples of preferred CTP's include themaize RUBISCO SSU protein CTP, and functionally related peptides such asthe Arabidopsis thaliana RUBISCO small subunit CTP and the Arabidopsisthaliana EPSPS CTP. These CTP's are exemplified by the polynucleotideand amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, and SEQ ID NO:14 respectively.

Recombinant plants, cells, seeds, and other plant tissues could also beproduced in which only the mitochondrial or chloroplast DNA has beenaltered to incorporate the molecules envisioned in this application.Promoters which function in chloroplasts have been known in the art(Hanley-Bowden et al., Trends in Biochemical Sciences 12:67-70, 1987).Methods and compositions for obtaining cells containing chloroplastsinto which heterologous DNA has been inserted has been described in U.S.Pat. No. 5,693,507 (1997). McBride et al. (WO 95/24492) discloselocalization and expression of genes encoding Cry1A δ-endotoxin proteinin tobacco plant chloroplast genomes.

An exemplary embodiment of the invention involves the plastid orchloroplast targeting or plastid or chloroplast localization of genesencoding enzymes or proteins conferring herbicide tolerance or herbicideresistance in plants. Plastid or chloroplast targeting sequences havebeen isolated from numerous nuclear encoded plant genes and have beenshown to direct importation of cytoplasmically synthesized proteins intoplastids or chloroplasts (reviewed in Keegstra and Olsen, 1989). Avariety of plastid targeting sequences, well known in the art, includingbut not limited to ADPGPP, EPSP synthase, or ssRUBISCO, may be utilizedin practicing this invention. In addition, plastidic targeting sequences(peptide and nucleic acid) for monocotyledonous crops may consist of agenomic coding fragment containing an intron sequence as well as aduplicated proteolytic cleavage site in the encoded plastidic targetingsequences.

The preferred CTP sequence for dicotyledonous crops is referred toherein as (SEQ ID NO:9), and consists of a genomic coding fragmentcontaining the chloroplast targeting peptide sequence from the EPSPsynthase gene of Arabidopsis thaliana in which the transit peptidecleavage site of the pea ssRUBISCO CTP replaces the native EPSP synthaseCTP cleavage site (Klee et al., 1987).

For optimized expression in monocotyledonous plants, an intron may alsobe included in the DNA expression construct. Such an intron is typicallyplaced near the 5′ end of the mRNA in untranslated sequence. This introncould be obtained from, but not limited to, a set of introns consistingof the maize heat shock protein (HSP) 70 intron (U.S. Pat. No.5,424,412; 1995), the rice Act1 intron (McElroy et al., 1990), the Adhintron 1 (Callis et al., 1987), or the sucrose synthase intron (Vasil etal., 1989).

The 3′ non-translated region of the genes of the present invention whichare localized to the plant nuclear genome also contain a polyadenylationsignal which functions in plants to cause the addition of adenylatenucleotides to the 3′ end of the mRNA. RNA polymerase transcribes anuclear genome coding DNA sequence through a site where polyadenylationoccurs. Typically, DNA sequences located a few hundred base pairsdownstream of the polyadenylation site serve to terminate transcription.Those DNA sequences are referred to herein as transcription-terminationregions. Those regions are required for efficient polyadenylation oftranscribed messenger RNA (mRNA). Examples of preferred 3′ regions are(1) the 3′ transcribed, non-translated regions containing thepolyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmidgenes, such as the nopaline synthase (NOS) gene and (2) the 3′ ends ofplant genes such as the pea ribulose-1,5-bisphosphate carboxylase smallsubunit gene, designated herein as E9 (Fischhoff et al., 1987).Constructs will typically include the gene of interest along with a 3′end DNA sequence that acts as a signal to terminate transcription and,in constructs intended for nuclear genome expression, allow for thepoly-adenylation of the resultant mRNA. The most preferred 3′ elementsare contemplated to be those from the nopaline synthase gene of A.tumefaciens (nos 3′ end) (Bevan et al., 1983), the terminator for the T7transcript from the octopine synthase gene of A. tumefaciens, and the 3′end of the protease inhibitor I or II genes from potato or tomato.Regulatory elements such as TMV Ω element (Gallie, et al., 1989), mayfurther be included where desired.

According to the present invention and as noted above, chloroplast orplastid localized genes encoding enzymes conferring herbicide toleranceor herbicide resistance characteristics to plants do not requiresequences which confer transcription termination and polyadenylationsignals, but instead may only require transcription terminationinformation at the 3′ end of the gene. For coding sequences introducedinto a chloroplast or plastid, or into a chloroplast or plastid genome,mRNA transcription termination is similar to methods well known in thebacterial gene expression art. For example, either in a polycistronic ora monocistronic sequence, transcription can be terminated by stem andloop structures or by structures similar to rho dependent sequences.

Transcription enhancers or duplications of enhancers could be used toincrease expression. These enhancers often are found 5′ to the start oftranscription in a promoter that functions in eukaryotic cells, but canoften be inserted in the forward or reverse orientation 5′ or 3′ to thecoding sequence. Examples of enhancers include elements from the CaMV35S promoter, octopine synthase genes (Ellis et al., 1987), the riceactin gene, and promoter from non-plant eukaryotes (e.g., yeast; Ma etal., 1988).

In certain embodiments of the invention, the use of internal ribosomebinding sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

Constructs intended for expression from within a chloroplast or plastidutilizing chloroplast or plastid specific transcriptional andtranslational machinery can contain either mono- or polycistronicsequences.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the polypeptide coding regionto which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of A. tumefaciens described (Rogers et al., 1987). However,several other plant integrating vector systems are known to function inplants including pCaMVCN transfer control vector described (Fromm etal., 1985). pCaMVCN (available from Pharmacia, Piscataway, N.J.)includes the CaMV35S promoter.

In preferred embodiments, the vector used to express the polypeptideincludes a selection marker that is effective in a plant cell,preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e. the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ non-translated region described (Rogers et al., 1988).

Means for preparing expression vectors are well known in the art.Expression (transformation) vectors used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011 (each of which is specificallyincorporated herein by reference). Those vectors can be modified toinclude a coding sequence in accordance with the present invention.

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to conferenhanced herbicide resistance enzymatic activity to a cell is preferablya polynucleotide encoding an AMPA transacylase or a functionalequivalent alone or in combination with a gene encoding a GOX enzyme ora functional equivalent of GOX. In accordance with such embodiments, acoding region comprising the DNA sequence of SEQ ID NO:3, SEQ ID NO:7,or SEQ ID NO:19 is also preferred.

Specific genes encoding AMPA transacylase that have been shown tosuccessfully transform plants in conjunction with plastid targetingpeptide-encoding genes, to express the AMPA transacylase at sufficientherbicidally protective levels are those genes comprised within theplasmid vectors. Preferred plasmids containing plastid targetingsequences include pMON17261, pMON10151, pMON10149, pMON32570, pMON32571,pMON32572, pMON32573, pMON32926, pMON32931, pMON32932, pMON32936,pMON32938, pMON32946, pMON32947, pMON32948, and pMON32950. Theseplasmids contain polynucleotide sequences which encode targetingsequences as shown in SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ IDNO:14. Expression cassettes comprising plant operable promoters linkedto coding sequences, some with and some without f′ untranslatedsequences and/or intron sequences, wherein the coding sequences containat least an AMPA transacylase or transacetylase, linked to plantoperable termination sequences are disclosed in particular as set forthin SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, and SEQ IDNO:31.

The work described herein has identified methods of potentiating inplanta expression of an AMPA transacylase, which confer protection fromglyphosate and related herbicides to plants when incorporated into thenuclear, plastid, or chloroplast genome of susceptible plants which alsoexpress a GOX or similar gene. U.S. Pat. No. 5,500,365 (specificallyincorporated herein by reference) describes a method for synthesizingplant genes to optimize the expression level of the protein for whichthe synthesized gene encodes. This method relates to the modification ofthe structural gene sequences of the exogenous transgene, to make themmore “plant-like” and therefore more likely to be translated andexpressed by the plant. A similar method for enhanced expression oftransgenes, preferably in monocotyledonous plants, is disclosed in U.S.Pat. No. 5,689,052 (specifically incorporated herein by reference).Agronomic, horticultural, ornamental, and other economically orcommercially useful plants can be made in accordance with the methodsdescribed herein.

Such plants may co-express the AMPA transacylase gene and/or a GOX genealong with other antifungal, antibacterial, or antiviralpathogenesis-related peptides, polypeptides, or proteins; insecticidalproteins; other proteins conferring herbicide resistance; and proteinsinvolved in improving the quality of plant products or agronomicperformance of plants. Simultaneous co-expression of multipleheterologous proteins in plants is advantageous in that it can exploitsmore than one mode of action to control plant damage or improve thequality of the plant or products produced by the plants metabolism.

It is contemplated that introduction of large DNA sequences comprisingmore than one gene may be desirable. Introduction of such sequences maybe facilitated by use of bacterial or yeast artificial chromosomes (BACsor YACs, respectively), or even plant artificial chromosomes. Forexample, the use of BACs for Agrobacterium-mediated transformation wasdisclosed by Hamilton et al. (1996).

Ultimately, the most desirable DNA sequences for introduction into amonocot genome may be homologous genes or gene families which encode adesired trait (for example, increased yield), and which are introducedunder the control of novel promoters or enhancers, etc., or perhaps evenhomologous or tissue specific (e.g., root-collar/sheath-, whorl-,stalk-, earshank-, kernel- or leaf-specific) promoters or controlelements. Indeed, it is envisioned that a particular use of the presentinvention may be the production of transformants comprising a transgenewhich is targeted in a tissue-specific manner. For example, herbicideresistance or herbicide tolerance genes may be expressed specifically orspecifically regulated in a negative manner in the plants reproductivetissues which can provide a means for enhancing herbicide tolerance orsensitivity to those tissues. Such regulatory control means can providemethods for regulating the escape of transgenes into the environment orfor controlling the illicit use of proprietary or licensed intellectualor commercialized property.

Vectors for use in tissue-specific targeting of gene expression intransgenic plants typically will include tissue-specific promoters andalso may include other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure.

It also is contemplated that tissue specific expression may befunctionally accomplished by introducing a constitutively expressed gene(all tissues) in combination with an antisense gene that is expressedonly in those tissues where the gene product is not desired. Forexample, a gene coding for the AMPA transacylase from E. coli may beintroduced such that it is expressed in all tissues using the 35Spromoter from Cauliflower Mosaic Virus. Alternatively, a rice actinpromoter or a histone promoter from a dicot or monocot species alsocould be used for constitutive expression of a gene. Furthermore, it iscontemplated that promoters combining elements from more than onepromoter may be useful. For example, U.S. Pat. No. 5,491,288 disclosescombining a Cauliflower Mosaic Virus promoter with a histone promoter.Therefore, expression of an antisense transcript of the AMPAtransacylase gene in a maize kernel, using for example a zein promoter,would prevent accumulation of the transacylase in seed. Thus, in a plantexpressing both GOX and the transacylase, application of glyphosateherbicide would result in seed tissues which fail to mature. Conversely,antisense suppression of the GOX gene would effectuate the same result.Preferably, suppression of the transacylase in specific tissues would bemore advantageous, particularly where specific tissues have demonstratedan intolerance to AMPA or related compounds. It is specificallycontemplated by the inventor that a similar strategy could be used withthe instant invention to direct expression of a screenable or selectablemarker in seed tissue.

Alternatively, one may wish to obtain novel tissue-specific promotersequences for use in accordance with the present invention. To achievethis, one may first isolate cDNA clones from the tissue concerned andidentify those clones which are expressed specifically in that tissue,for example, using Northern blotting. Ideally, one would like toidentify a gene that is not present in a high copy number, but whichgene product is relatively abundant in specific tissues. The promoterand control elements of corresponding genomic clones may this belocalized using the techniques of molecular biology known to those ofskill in the art.

It is contemplated that expression of some genes in transgenic plantswill be desired only under specified conditions. For example, it isproposed that expression of certain genes that confer resistance toenvironmentally stress factors such as drought will be desired onlyunder actual stress conditions. It further is contemplated thatexpression of such genes throughout a plants development may havedetrimental effects. It is known that a large number of genes exist thatrespond to the environment. For example, expression of some genes suchas rbcS, encoding the small subunit of ribulose bisphosphatecarboxylase, is regulated by light as mediated through phytochrome.Other genes are induced by secondary stimuli. For example, synthesis ofabscisic acid (ABA) is induced by certain environmental factors,including but not limited to water stress. A number of genes have beenshown to be induced by ABA (Skriver and Mundy, 1990). It also isexpected that expression of genes conferring resistance to applicationsof herbicides would be desired only under conditions in which herbicideis actually present. Therefore, for some desired traits, inducibleexpression of genes in transgenic plants will be desired.

It is proposed that, in some embodiments of the present invention,expression of a gene in a transgenic plant will be desired only in acertain time period during the development of the plant. Developmentaltiming frequently is correlated with tissue specific gene expression.For example expression of zein storage proteins is initiated in theendosperm about 15 days after pollination.

It also is contemplated that it may be useful to specifically target DNAinsertion within a cell. For example, it may be useful to targetintroduced DNA to the nucleus, and in particular into a precise positionwithin one of the plant chromosomes in order to achieve site specificintegration. For example, it would be useful to have a gene introducedthrough transformation which acts to replace an existing gene in thecell, or to complement a gene which is not functional or present at all.

A plant transformed with an expression vector of the present inventionis also contemplated. A transgenic plant derived from such a transformedor transgenic cell is also contemplated. Those skilled in the art willrecognize that a chimeric plant gene containing a structural codingsequence of the present invention can be inserted into the genome of aplant by methods well known in the art. Such methods for DNAtransformation of plant cells include Agrobacterium-mediated planttransformation, the use of liposomes, transformation using viruses orpollen, electroporation, protoplast transformation, gene transfer intopollen, injection into reproductive organs, injection into immatureembryos and particle bombardment. Each of these methods has distinctadvantages and disadvantages. Thus, one particular method of introducinggenes into a particular plant strain may not necessarily be the mosteffective for another plant strain, but it is well known which methodsare useful for a particular plant strain.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as infection by A. tumefaciensand related Agrobacterium strains, direct delivery of DNA such as, forexample, by PEG-mediated transformation of protoplasts (Omirulleh etal., 1993), by desiccation/inhibition-mediated DNA uptake, byelectroporation, by agitation with silicon carbide fibers, byacceleration of DNA coated particles, etc. In certain embodiments,acceleration methods are preferred and include, for example,microprojectile bombardment and the like.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973);(2) physical methods such as microinjection (Capecchi, 1980),electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and thegene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) viralvectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a;1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992;Wagner et al., 1992).

The application of brief, high-voltage electric pulses to a variety ofanimal and plant cells leads to the formation of nanometer-sized poresin the plasma membrane. DNA is taken directly into the cell cytoplasmeither through these pores or as a consequence of the redistribution ofmembrane components that accompanies closure of the pores.Electroporation can be extremely efficient and can be used both fortransient expression of cloned genes and for establishment of cell linesthat carry integrated copies of the gene of interest. Electroporation,in contrast to calcium phosphate-mediated transfection and protoplastfusion, frequently gives rise to cell lines that carry one, or at most afew, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation is well-known tothose of skill in the art. To effect transformation by electroporation,one may employ either friable tissues such as a suspension culture ofcells, or embryogenic callus, or alternatively, one may transformimmature embryos or other organized tissues directly. One wouldpartially degrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wounding in acontrolled manner, rendering the cells more susceptible totransformation. Such cells would then be recipient to DNA transfer byelectroporation, which may be carried out at this stage, and transformedcells then identified by a suitable selection or screening protocoldependent on the nature of the newly incorporated DNA.

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. Using these particles, DNA iscarried through the cell wall and into the cytoplasm on the surface ofsmall metal particles as described (Klein et al., 1987; Klein et al.,1988; Kawata et al., 1988). The metal particles penetrate throughseveral layers of cells and thus allow the transformation of cellswithin tissue explants. The microprojectile bombardment method ispreferred for the identification of chloroplast or plastid directedtransformation events.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming plant cells, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into plant cells byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered with theplant cultured cells in suspension. The screen disperses the particlesso that they are not delivered to the recipient cells in largeaggregates. It is believed that a screen intervening between theprojectile apparatus and the cells to be bombarded reduces the size ofprojectiles aggregate and may contribute to a higher frequency oftransformation by reducing damage inflicted on the recipient cells byprojectiles that are too large.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themicroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature plant embryos.

Accordingly, it is contemplated that one may desire to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art in light of the presentdisclosure.

The methods of particle-mediated transformation is well-known to thoseof skill in the art. U.S. Pat. No. 5,015,580 (specifically incorporatedherein by reference) describes the transformation of soybeans using sucha technique.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). The genetic engineering of cottonplants using Agrobacterium-mediated transfer is described in U.S. Pat.No. 5,004,863 (specifically incorporated herein by reference); liketransformation of lettuce plants is described in U.S. Pat. No. 5,349,124(specifically incorporated herein by reference); and theAgrobacterium-mediated transformation of soybean is described in U.S.Pat. No. 5,416,011 (specifically incorporated herein by reference).Further, the integration of the Ti-DNA is a relatively precise processresulting in few rearrangements. The region of DNA to be transferred isdefined by the border sequences, and intervening DNA is usually insertedinto the plant genome as described (Spielmann et al., 1986; Jorgensen etal., 1987).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus using Agrobacterium vectors as described (Bytebieret al., 1987). Other monocots recently have also been transformed withAgrobacterium. Included in this group are corn (Ishida et al.) and rice(Cheng et al.).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene.However, inasmuch as use of the word “heterozygous” usually implies thepresence of a complementary gene at the same locus of the secondchromosome of a pair of chromosomes, and there is no such gene in aplant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

An independent segregant may be preferred when the plant iscommercialized as a hybrid, such as corn. In this case, an independentsegregant containing the gene is crossed with another plant, to form ahybrid plant that is heterozygous for the gene of interest.

An alternate preference is for a transgenic plant that is homozygous forthe added structural gene; i.e. a transgenic plant that contains twoadded genes, one gene at the same locus on each chromosome of achromosome pair. A homozygous transgenic plant can be obtained bysexually mating (selfing) an independent segregant transgenic plant thatcontains a single added gene, germinating some of the seed produced andanalyzing the resulting plants produced for gene of interest activityand mendelian inheritance indicating homozygosity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

Two different transgenic plants can be mated to produce offspring thatcontain two independently segregating added, exogenous genes. Selfing ofappropriate progeny can produce plants that are homozygous for bothadded, exogenous genes that encode a polypeptide of interest.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant germplasm depends uponthe ability to regenerate that particular plant variety fromprotoplasts. Illustrative methods for the regeneration of cereals fromprotoplasts are described (see, e.g., Fujimura et al., 1985; Toriyama etal., 1986; Yamada et al., 1986; Abdullah et al., 1986).

To transform plant germplasm that cannot be successfully regeneratedfrom protoplasts, other ways to introduce DNA into intact cells ortissues can be utilized. For example, regeneration of cereals fromimmature embryos or explants can be effected as described (Vasil, 1988).

Unmodified bacterial genes are often poorly expressed in transgenicplant cells. Plant codon usage more closely resembles that of humans andother higher organisms than unicellular organisms, such as bacteria.Several reports have disclosed methods for improving expression ofrecombinant genes in plants (Murray et al., 1989; Diehn et al., 1996;Iannacone et al., 1997; Rouwendal et al., 1997; Futterer et al., 1997;and Futterer and Hohn, 1996). These reports disclose various methods forengineering coding sequences to represent sequences which are moreefficiently translated based on plant codon frequency tables,improvements in codon third base position bias, using recombinantsequences which avoid suspect polyadenylation or A/T rich domains orintron splicing consensus sequences.

U.S. Pat. No. 5,500,365 (specifically incorporated herein by reference)describes the preferred method for synthesizing plant genes to optimizethe expression level of the protein for which the synthesized geneencodes. This method relates to the modification of the structural genesequences of the exogenous transgene, to make them more “plant-like” andtherefore more likely to be translated and expressed by the plant,monocot or dicot. However, the method as disclosed in U.S. Pat. No.5,689,052 provides for enhanced expression of transgenes, preferably inmonocotyledonous plants, which is herein incorporated in its entirety byreference. Briefly, according to Brown et al., the frequency of rare andsemi-rare monocotyledonous codons in a polynucleotide sequence encodinga desired protein are reduced and replaced with more preferredmonocotyledonous codons. Enhanced accumulation of a desired polypeptideencoded by a modified polynucleotide sequence in a monocotyledonousplant is the result of increasing the frequency of preferred codons byanalyzing the coding sequence in successive six nucleotide fragments andaltering the sequence based on the frequency of appearance of thesix-mers as to the frequency of appearance of the rarest 284, 484, and664 six-mers in monocotyledenous plants. Furthermore, Brown et al.disclose the enhanced expression of a recombinant gene by applying themethod for reducing the frequency of rare codons with methods forreducing the occurrence of polyadenylation signals and intron splicesites in the nucleotide sequence, removing self-complementary sequencesin the nucleotide sequence and replacing such sequences withnonself-complementary nucleotides while maintaining a structural geneencoding the polypeptide, and reducing the frequency of occurrence of5′-CG-3′ di-nucleotide pairs in the nucleotide sequence. These steps areperformed sequentially and have a cumulative effect resulting in anucleotide sequence containing a preferential utilization of themore-preferred monocotyledonous codons for monocotyledonous plants for amajority of the amino acids present in the desired polypeptide.

Thus, the amount of a gene coding for a polypeptide of interest can beincreased in plants by transforming those plants using transformationmethods such as those disclosed herein. In particular, chloroplast orplastid transformation can result in desired coding sequences beingpresent in up to about 10,000 copies per cell in tissues containingthese subcellular organelle structures (McBride et al., Bio/Technology13:362-365, 1995).

DNA can also be introduced into plants by direct DNA transfer intopollen as described (Zhou et al., 1983; Hess, 1987). Expression ofpolypeptide coding genes can be obtained by injection of the DNA intoreproductive organs of a plant as described (Pena et al., 1987). DNA canalso be injected directly into the cells of immature embryos andintroduced into cells by rehydration of desiccated embryos as described(Neuhaus et al., 1987; Benbrook et al., 1986).

After effecting delivery of exogenous DNA to recipient cells, the nextstep to obtain a transgenic plant generally concern identifying thetransformed cells for further culturing and plant regeneration. Asmentioned herein, in order to improve the ability to identifytransformants, one may desire to employ a selectable or screenablemarker gene as, or in addition to, the expressible gene of interest. Inthis case, one would then generally assay the potentially transformedcell population by exposing the cells to a selective agent or agents, orone would screen the cells for the desired marker gene trait.

An exemplary embodiment of methods for identifying transformed cellsinvolves exposing the transformed cultures to a selective agent, such asa metabolic inhibitor, an antibiotic, herbicide or the like. Cells whichhave been transformed and have stably integrated a marker geneconferring resistance to the selective agent used, will grow and dividein culture. Sensitive cells will not be amenable to further culturing.One example of a preferred marker gene confers resistance to glyphosate.When this gene is used as a selectable marker, the putativelytransformed cell culture is treated with glyphosate. Upon treatment,transgenic cells will be available for further culturing whilesensitive, or non-transformed cells, will not. This method is describedin detail in U.S. Pat. No. 5,569,834, which is specifically incorporatedherein by reference. Another example of a preferred selectable markersystem is the neomycin phosphotransferase (nptII) resistance system bywhich resistance to the antibiotic kanamycin is conferred, as describedin U.S. Pat. No. 5,569,834 (specifically incorporated herein byreference). Again, after transformation with this system, transformedcells will be available for further culturing upon treatment withkanamycin, while non-transformed cells will not. Yet another preferredselectable marker system involves the use of a gene construct conferringresistance to paromomycin. Use of this type of a selectable markersystem is described in U.S. Pat. No. 5,424,412 (specificallyincorporated herein by reference).

Another preferred selectable marker system involves the use of the genescontemplated by this invention. In particular, a phnO gene or asubstantially similar gene encoding an AMPA transacylase can be utilizedas a selectable marker. Plant cells which have had a recombinant DNAmolecule introduced into their genome can be selected from a populationof cells which failed to incorporate a recombinant molecule by growingthe cells in the presence of AMPA. One skilled in the art will recognizethe particular advantages that this selectable marker system has overprevious selectable marker systems. The selectable marker used in therecombinant DNA integrated into a plant genome reduces the amount of DNAtargeted for integration because the selectable marker will also be usedfor improved herbicide tolerance or improved herbicide resistance inplants generated from transformed plant cells. This selectable markeralso provides an additional marker system not known before, particularlyin a field in which there are often only a limited number of selectablemarkers available.

Transplastonomic selection (selection of plastid or chloroplasttransformation events) is simplified by taking advantage of thesensitivity of chloroplasts or plastids to spectinomycin, an inhibitorof plastid or chloroplast protein synthesis, but not of proteinsynthesis by the nuclear genome encoded cytoplasmic ribosomes.Spectinomycin prevents the accumulation of chloroplast proteins requiredfor photosynthesis and so spectinomycin resistant transformed plantcells may be distinguished on the basis of their difference in color:the resistant, transformed cells are green, whereas the sensitive cellsare white, due to inhibition of plastid-protein synthesis.Transformation of chloroplasts or plastids with a suitable bacterial aadgene, or with a gene encoding a spectinomycin resistant plastid orchloroplast functional ribosomal RNA provides a means for selection andmaintenance of transplastonomic events (Maliga, Trends in Biotechnology11:101-106, 1993).

It is further contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such asglyphosate or kanamycin, may either not provide enough killing activityto clearly recognize transformed cells or may cause substantialnonselective inhibition of transformants and nontransformants alike,thus causing the selection technique to not be effective. It is proposedthat selection with a growth inhibiting compound, such as glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as kanamycin would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. It is proposedthat combinations of selection and screening may enable one to identifytransformants in a wider variety of cell and tissue types. Theavailability of the transacylases of the present invention may obviatethe necessity for combination selection and screening by providing anadditional selection means.

The development or regeneration of plants from either single plantprotoplasts or various explants is well known in the art (Weissbach andWeissbach, 1988). This regeneration and growth process typicallyincludes the steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983). In particular, U.S. Pat.No. 5,349,124 (specification incorporated herein by reference) detailsthe creation of genetically transformed lettuce cells and plantsresulting therefrom which express hybrid crystal proteins conferringinsecticidal activity against Lepidopteran larvae to such plants.

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, or pollen obtained from the regeneratedplants is crossed to seed-grown plants of agronomically important,preferably inbred lines. Conversely, pollen from plants of thoseimportant lines is used to pollinate regenerated plants. A transgenicplant of the present invention containing a desired polypeptide iscultivated using methods well known to one skilled in the art.

In one embodiment, a transgenic plant of this invention thus has anincreased amount of a coding region encoding an AMPA transacylasepolypeptide which may also be expressed along with a plastid targetingpeptide. A preferred transgenic plant is an independent segregant andcan transmit that gene and its activity to its progeny. A more preferredtransgenic plant is homozygous for that gene, and transmits that gene toall of its offspring on sexual mating. Seed from a transgenic plant maybe grown in the field or greenhouse, and resulting sexually maturetransgenic plants are self-pollinated to generate true breeding plants.The progeny from these plants become true breeding lines that areevaluated for expression of the transacylase transgene as well as forimproved herbicide tolerance, particularly when the transacylasetransgene is co-expressed along with a gene encoding a GOX enzyme.

The genes and acyltransferases according to the subject inventioninclude not only the full length sequences disclosed herein but alsofragments of these sequences, or fusion proteins, which retain thecharacteristic improved herbicidal protective activity of the sequencesspecifically exemplified herein.

It should be apparent to a person of skill in this art that AMPAtransacylase genes and peptides can be identified and obtained throughseveral means. The specific genes, or portions thereof, may be obtainedfrom a culture depository, or constructed synthetically, for example, byuse of a gene machine. Variations of these genes may be readilyconstructed using standard techniques for making point mutations. Also,fragments of these genes can be made using commercially availableexonucleases or endonucleases according to standard procedures. Forexample, enzymes such as Bal31 or site-directed mutagenesis can be usedto systematically cut off nucleotides from the ends of these genes.Also, genes which code for active fragments may be obtained using avariety of other restriction enzymes. Proteases may be used to directlyobtain active fragments of such transacylases.

Equivalent AMPA transacylases and/or genes encoding these transacylasescan also be isolated from E. coli strains and/or DNA libraries using theteachings provided herein. For example, antibodies to the transacylasesdisclosed and claimed herein can be used to identify and isolate othertransacylases from a mixture of proteins. Specifically, antibodies maybe raised to the transacylases disclosed herein and used to specificallyidentify equivalent AMPA transacylases by immunoprecipitation, columnimmuno-purification, enzyme linked immunoassay (ELISA), or Westernblotting.

A further method for identifying the peptides and genes of the subjectinvention is through the use of oligonucleotide probes. These probes arenucleotide sequences having a detectable label. As is well known in theart, if the probe molecule and sequences in a target nucleic acid samplehybridize by forming a strong bond between the two molecules, it can bereasonably assumed that the probe and target sample contain essentiallyidentical polynucleotide sequences. The probe's detectable labelprovides a means for determining in a known manner whether hybridizationhas occurred. Such a probe analysis provides a rapid method foridentifying AMPA transacylase genes of the subject invention.

The nucleotide segments which are used as probes according to theinvention can be synthesized by use of DNA synthesizers using standardprocedures. In the use of the nucleotide segments as probes, theparticular probe is labeled with any suitable label known to thoseskilled in the art, including radioactive and non-radioactive labels.Typical radioactive labels include ³²P, ¹²⁵I, ³⁵S, or the like. A probelabeled with a radioactive isotope can be constructed from a nucleotidesequence complementary to the DNA sample by a conventional nicktranslation reaction, using a DNase and DNA polymerase. The probe andsample can then be combined in a hybridization buffer solution and heldat an appropriate temperature until annealing occurs. Thereafter, themembrane is washed free of extraneous materials, leaving the sample andbound probe molecules typically detected and quantified byautoradiography and/or liquid scintillation counting.

Non-radioactive labels include, for example, ligands such as biotin orthyroxin, as well as enzymes such as hydrolyses or peroxidases, or thevarious chemiluminescers such as luciferin, or fluorescent compoundslike fluorescein, rhodamine, Texas Red, and derivatives and the like.The probe may also be labeled at both ends with different types oflabels for ease of separation, as, for example, by using an isotopiclabel at the end mentioned above and a biotin label at the other end, orwith different fluorescent emitters which have overlapping absorptionand emission spectra.

Duplex formation and stability depend on substantial complementarybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probes of thesubject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, by methods currently known to anordinarily skilled artisan, and perhaps by other methods which maybecome known in the future.

The potential variations in the probes listed is due, in part, to theredundancy of the genetic code. Because of the redundancy of the geneticcode, more than one coding nucleotide triplet (codon) can be used formost of the amino acids used to make proteins. Therefore differentnucleotide sequences can code for a particular amino acid. Thus, theamino acid sequence of the E. coli AMPA transacylase and peptide, andthe plastid targeting peptides and the polynucleotides which code forthem, can be prepared by equivalent nucleotide sequences encoding thesame amino acid sequence of the protein or peptide. Accordingly, thesubject invention includes such equivalent nucleotide sequences. Also,inverse or complement sequences are an aspect of the subject inventionand can be readily used by a person skilled in this art. In addition ithas been shown that proteins of identified structure and function may beconstructed by changing the amino acid sequence if such changes do notalter the protein secondary structure (Kaiser and Kezdy, 1984). Thus,the subject invention includes mutants of the amino acid sequencedepicted herein which do not alter the protein secondary structure, orif the structure is altered, the biological activity is substantiallyretained. Further, the invention also includes mutants of organismshosting all or part of a gene encoding an AMPA acyltransferase and/orgene encoding a plastid targeting peptide, as discussed in the presentinvention. Such mutants can be made by techniques well known to personsskilled in the art. For example, UV irradiation can be used to preparemutants of host organisms. Likewise, such mutants may includeasporogenous host cells which also can be prepared by procedures wellknown in the art.

Site-specific or site-directed mutagenesis is a technique useful in thepreparation of individual, novel and unique useful peptides, orbiologically functional equivalent proteins or peptides, throughspecific mutagenesis of structural genes encoding such peptides. Thetechnique further provides a ready ability to prepare and test sequencevariants by altering the coding sequence of a gene, for example, byintroducing one or more nucleotide sequence changes into the DNA for thepurpose of creating a new or useful restriction endonuclease cleavagerecognition sequence or for the purpose of altering the coding sequenceso that a gene's codons and percent G/C represent those more commonlyused by a particular genus or species. Site-specific mutagenesis allowsthe production of deletion, insertion, or replacement mutations throughthe use of specific mutagenesis oligonucleotide sequences comprising theDNA sequence of the desired mutation. Mutagenesis oligonucleotidestypically provide a primer sequence of sufficient size and sequencecomplexity to form a stable duplex on both sides of the desired mutationtarget site. Typically, a primer of about 17 to 25 nucleotides in lengthis preferred, with about 5 to 10 residues overlapping either side of thedesired mutation target site.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis, and often contain afilamentous phage origin of replication which, in the presence of ahelper phage, allows synthesis of single stranded DNA from the plasmidvector.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a mutation target site. A mutagenesis oligonucleotide primerbearing the desired mutant sequence is prepared, generallysynthetically. The mutagenesis primer is then annealed with thesingle-stranded vector at the mutation target site, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected which include recombinantvectors containing the mutation represented by the mutagenesis primersequence.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Such procedures may favorably change the protein's biochemical andbiophysical characteristics or its mode of action. These include, butare not limited to: 1) improved AMPA transacylase formation, 2) improvedprotein stability or reduced protease degradation, 3) improved substraterecognition and binding, 4) improved enzyme kinetics, and 5) improvedN-acyl-AMPA formation due to any or all of the reasons stated above.

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The biologically functional equivalentpeptides, polypeptides, and proteins contemplated herein should possessat least from about 40% to about 65% sequence similarity, preferablyfrom about 66% to about 75% sequence similarity, more preferably fromabout 76% to about 85% similarity, and most preferably from about 86% toabout 90% or greater sequence similarity to the sequence of, orcorresponding moiety within, the AMPA acyltransferase amino acidsequences disclosed herein.

The following is a discussion based upon changing the amino acids of aprotein to create an equivalent, or even an improved, second-generationmolecule. In particular embodiments of the invention, mutated AMPAtransacylase proteins are contemplated to be useful for improving orenhancing the in planta expression of the protein, and consequentlyincreasing or improving the AMPA transacylase activity and/or expressionof the recombinant transgene in a plant cell. The amino acid changes maybe achieved by changing the codons of the DNA sequence, according to thecodons given in Table 1, in dicotyledonous, and more particularly inmonocotyledonous plants.

TABLE 1 Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventor that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Polynucleotides encoding heterologous proteins are known by thoseskilled in the art, to often be poorly expressed when incorporated intothe nuclear DNA of transgenic plants (reviewed by Diehn et al., 1996).Preferably, a nucleotide sequence encoding a heterologous protein ofinterest is designed essentially as described in U.S. Pat. Nos.5,500,365 and 5,689,052 (each specifically incorporated herein byreference). Examples of nucleotide sequences useful for expressioninclude but are not limited to, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11,and SEQ ID NO:19.

Substitutes for an amino acid within the fundamental polypeptidesequence can be selected from other members of the class to which thenaturally occurring amino acid belongs. Amino acids can be divided intothe following four groups: (1) acidic amino acids; (2) basic aminoacids; (3) neutral polar amino acids; and (4) neutral non-polar aminoacids. Representative amino acids within these various groups include,but are not limited to: (1) acidic (negatively charged) amino acids suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids such as arginine, histidine, and lysine; (3) neutral polar aminoacids such as glycine, serine, threonine, cyteine, cystine, tyrosine,asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine.

Conservative amino acid changes within a fundamental polypeptidesequence can be made by substituting one amino acid within one of thesegroups with another amino acid within the same group. The encodingnucleotide sequence (gene, plasmid DNA, cDNA, or synthetic DNA) willthus have corresponding base substitutions, permitting it to encodebiologically functional equivalent forms of an AMPA transacylase.

The following examples describe preferred embodiments of the invention.Other embodiments within the scope of the claims herein will be apparentto one skilled in the art of endeavor from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples. In the examplesall percentages are given on a weight basis unless otherwise indicated.

EXAMPLES Example 1

This example illustrates the growth inhibitory effects of N-aminomethylphosphonic acid (AMPA) on plant callus tissue, and the lack ofinhibition of N-acetyl-aminomethyl phosphonic acid on plant callustissue in in vitro culture conditions.

Certain recombinant plant species which express a bacterial GOX gene,and which were also exposed to glyphosate, can exhibit phytotoxiceffects manifested through such symptoms as chlorosis, flowerabscission, and reduced fertility. The basis for these symptoms had notpreviously been determined. Previous studies had indicated that plantsexpressing GOX metabolized glyphosate to AMPA and glyoxylate (U.S. Pat.No. 5,463,175). Glyoxylate is readily metabolized by plants, howeverAMPA persists in plant tissues and may be the cause of phytotoxiceffects such as chlorosis, stunting, or other undesirable effects. Ithad previously been shown that Achromobacter species LBAA was able toenzymatically modify AMPA to N-acetyl AMPA (U.S. Pat. No. 5,463,175).The Achromobacter data, coupled with the plant phytotoxicity data,indicated that N-acylation of AMPA in planta may provide effectiverelief from chlorosis and other undesirable effects. Thus, tobaccocallus tissue was exposed to AMPA and to N-acetyl AMPA in order todetermine if either of these compounds exhibited cytotoxic effectssimilar to those observed in plants expressing GOX and exposed toglyphosate.

Tobacco callus was generated from leaf pieces of wild type Nicotianatabacum cv. “Samsun” tobacco on MS104 plates (MS salts 4.3 g/l, sucrose30 g/l, B5 vitamins 500×2 ml/l, NAA 0.1 mg/l, and Bacto Agar 1.0 mg/l).Callus tissue was applied to plates with or without AMPA and with orwithout N-acetyl AMPA. Plates contained AMPA or N-acetyl AMPA atconcentrations of 0.1 mM or 0.4 mM. Plates were incubated for up tothree weeks and monitored periodically.

Callus tissue on control plates containing no AMPA or N-acetyl AMPA grewat normal rates, regenerating roots and shoots as expected. Callustissue in the presence of AMPA was severely inhibited. No growth wasobserved, showing the phytotoxic effect of AMPA at these concentrations.Callus tissue on plates containing N-acetyl AMPA was not inhibited, andformed roots and shoots similar to control callus tissue growth. Thisresult indicated that AMPA, as a byproduct of GOX mediated metabolism ofglyphosate, could be responsible for the observed phototoxicity inplants. This result also indicated the possibility of an improved methodfor selecting plants from genetically transformed callus tissue, as wellas a possible method for enhancing glyphosate herbicide resistance.

Example 2

This example illustrates that degradation of glyphosate by GOX enzymehydrolysis in the bacterium Achromobacter sp. strain LBAA results in theproduction of AMPA and N-acetyl AMPA.

It has been previously shown that GOX mediated glyphosate degradationproduced glyoxylate and AMPA (Barry et al., U.S. Pat. No. 5,463,175).Achromobacter sp. strain LBAA was also shown to produce AMPA andglyoxylate as a result of glyphosate degradation. The glyphosatedegradation pathway was characterized in resting cells ofglyphosate-grown Achromobacter sp. strain LBAA according to thefollowing procedure. Cells from a 100 ml culture of LBAA, grown in DF3Smedium containing glucose, gluconate and citrate as carbon sources andwith thiamine and Yeast Extract (0.01%) to supply trace requirements andwith glyphosate at 0.2 mM as a phosphorous source, were harvested at acell density of 200 Klett units, washed twice with 20 ml of DF3S mediumand the equivalent of 20 ml of cells were resuspended in 100 μl of thesame medium containing [¹⁴C]glyphosate (2.5 ml of 52 mCi/mmol, Amersham;CFA.745). The cell mix was incubated at 30° C. with shaking and 20 mlsamples were withdrawn at various intervals. The samples werecentrifuged to separate the cells from the broth supernatant. Both thesupernatant and cell pellets were analyzed by HPLC.

Samples prepared in this way were analyzed by strong anion exchange(SAX) HPLC with radioisotope label detection to determine their levelsof [¹⁴C]-AMPA and N-acetyl-[¹⁴C]-AMPA. Samples were injected using aWaters WISP autoinjector. Chromatographic profiles and quantitative datawere collected using MACS2, Monsanto's automated chromatography datacollection system. A Spherisorb S5 SAX, 250 mm×10 mm column, or anAlltech 5 micron, 250 mm×10 mm SAX column was used for the analyses.Solvents used were designated as solution A and solution B. Solution Acontained 0.005M KH₂PO₄ adjusted to pH 2.0 with H₃PO₄ in 4% methanol.Solution B contained 0.10 M KH₂PO₄ adjusted to pH 2.0 with H₃PO₄ in 4%methanol. Each sample run time consisted of a step gradient program withan eluent flow rate of 3 ml per minute and a scintillation fluid(tradename ATOMFLOW, No. NEN-995 obtained from Packard Instruments) flowrate of 9 ml per minute. The HPLC solvent profile for distinguishing[¹⁴C]-AMPA from N-acetyl-[¹⁴C]-AMPA in each sample analyzed wasrepresented by 100% solvent A at times zero through 5 minutes, thensolvent B at 100% at time 5 minutes through 15 minutes, then 100%solvent A through 20 minutes at which time the column is prepared toreceive another sample.

Cell pellets were first resuspended in DF3S medium made acidic byaddition of 0.65N HCl, boiled for 5 minutes, then centrifuged briefly toprovide a solution phase for HPLC analysis. Supernatants were treatedsimilarly prior to HPLC analysis. An acidified glyphosate control wasalso subjected to HPLC analysis, and the glyphosate retention time (RT)was determined to be 10.8 minutes. The amount of radioactivity in theglyphosate peak remaining in the supernatant after two hours incubationhad decreased to about 33% of the initial levels, indicating that theglyphosate was extensively metabolized. About 3% of the glyphosate wasfound to be within the cell. Material co-eluting with the methylaminestandard with an RT of 6 minutes accounted for about 5% of the initialamount of radioactivity in the supernatant and for about 1.5% of theinitial amount of radioactivity identified in the cell contents.

The GOX mediated glyphosate degradation pathway was elucidated furtherin a subsequent experiment where the metabolism of [¹⁴C]AMPA wascompared to that of [¹⁴C]glyphosate as indicated above in resting cellsharvested at 165 Klett units and resuspended at the equivalent of 15 mlcells per 100 ml DF3S medium. The samples were analyzed by HPLC andconsisted of whole cultures acidified and treated as described above.Cultures exposed to [¹⁴C]glyphosate for two hours were found to have 25%of the label in the methylamine/N-acetyl-methylamine peak with aretention time of 14.7 minutes, 12.5% as AMPA with a retention time of 6minutes, 30% in a peak with a retention time of 13.2 minutes, and 30% asglyphosate with a retention time of 10.8 minutes. Analysis of culturesexposed to [¹⁴C]-AMPA for two hours indicated that 15% of the label wasfound as N-acetyl-methylamine/methylamine, 59% as AMPA, and 18% in the13.2 minute peak. The material eluting at 13.2 minutes was identified asN-acetyl-AMPA by negative ion electrospray mass spectrometry. The resultshowed strong ions at m/e 152 and m/e 154, as expected for thiscompound, which has a molecular weight of 153 Daltons. The m/e 154 ionwas due to the isotopic ¹⁴C atom. N-acetyl-methyl-[¹⁴C]-AMPA arises fromN-methyl-[¹⁴C]-AMPA, which is a known impurity in preparations of[¹⁴C]-AMPA.

These data indicated that the glyphosate degradation pathway inAchromobacter strain LBAA proceeds from hydrolysis of glyphosate toAMPA, which is then converted to the products methylamine presumablythrough a dephosphorylation step, and N-acetyl-AMPA presumably throughsome previously unknown transacylation step. A small amount ofN-acetyl-AMPA is then converted to N-acetyl-methylamine. A similaracylation step has been inferred from the products identified in E. coliwhen aminomethylphosphonates are utilized as sole sources of phosphate(Avila et al., 1987).

Example 3

This example illustrates the identification of an AMPA acyltransferaseactivity in E. coli.

Avila et al. (1987) identified dephosphorylated biodegradation productsfrom the metabolism of a variety of aminophosphonate substrates used assole phosphate sources in vivo in E. coli while studying C—P bondscission. Their studies indicated that AMPA was not a substrate foracylation in E. coli K-12. In addition, Avila et al. were interested inthe effect of N-linked chemical substitutions on C—P bond scission ofphosphonates in E. coli, and identified N-acetylated products derivedfrom the metabolism of some aminophosphonates. Avila et al. alsodemonstrated that ‘wild type’ E. coli K12 strains, unlike wild type E.coli B strains, are unable to use phosphonates as a source of phosphate.Thus, in consideration of the phytotoxic effects of AMPA on callustissue as shown in Example 1 and the generation of AMPA from GOXmediated glyphosate degradation as shown in Example 2, the E. coli datain Avila et al. indicated that there may be an enzyme or pathway presentin some bacterial species which is capable of convertingaminomethylphosphonate (AMPA) to N-acetyl-AMPA. An enzyme or pathwaywith those characteristics would, if expressed in plants, confer asignificant advantage to plants expressing GOX when treated withglyphosate.

To test this, an E. coli K-12 strain adapted for growth on AMPA wasgrown on low phosphate containing medium in order to obtain cell lysatesto be assayed for the presence of an enzyme capable of AMPA N-acylation.The phn (mpu) operon is cryptic in E. coli K-12 due to an 8 base pairinsertion which causes a frameshift mutation in the phnE gene. Theframeshift inactivates PhnE and creates a polar effect on translation ofother genes downstream of phnE within the operon, resulting in theinability of such mutants to use phosphonates as phosphate sources(Makino et al., J. Bacteriol. 173:2665-2672, 1991). Selection of aspontaneously derived mutation restores the function of the phn operon(phn+ or mpu+). Thus, K-12 strains adapted for growth on AMPA,methyl-phosphonate, or ethyl-phosphonate contain such effectivespontaneously derived mutations.

Briefly, an aliquot of a fresh L-broth culture of E. coli K-12 strainJM101 (mpu−) was plated onto MOPS (Neidhardt et al., 1974) complete agarmedium containing amino acids at 25 mg/ml, vitamin B1 [thiamine] at 10mg/ml, 0.2% glucose, and 1.5% DIFCO “Purified” agar along withaminomethylphosphonate (AMPA; 0.2 mM; Sigma Chemical Co., St. Louis,Mo.) as the sole phosphate source, and incubated at 37° C. for threedays. Colonies arising on this media were picked and streaked onto MOPScomplete agar containing either AMPA or methylphosphonate (Alfa) as thesole phosphate source. One colony, designated E. coli JM101 mpu+, waschosen from those that grew equally and uniformly on both phosphonatecontaining media, and was further designated as E. coli strain GB993.

The phn operon is induced when E. coli is grown in media lacking orlimited in a phosphate source. Therefore, E. coli GB993 was compared tothe parental JM101 strain when grown in MOPS minimal media. GB993 andits mpu− parent strain, JM101, were grown under identical conditions,varying only the amount of phosphate available or supplemented withAMPA. 50 ml cultures were grown in duplicate in 250 mlsidearm-Erlenmeyer flasks with continuous shaking at 37° C. in MOPSmedium (5 mls of 10×MOPS salts, 0.5 ml 1 mg/ml thiamin, 0.5 ml 20%glucose, to 50 mls with dH₂0) containing 0.1 or 5 mM phosphate, or 0.1mM phosphate supplemented with approximately 0.2 mM AMPA, pH 7.0. Thecultures were generally grown to about 220 Klett units and the cellswere pelleted by centrifugation, resuspended in 1.5 mls of 10 mM Tris/1mM DTT, and lysed with two passes through a French press at 1,000 psi.Lysates were centrifuged to remove debris and the supernatant passedthrough a G-50 column equilibrated with 50 mM Tris pH 7.0. Table 2 showsthe results of cell cultures grown in this manner.

TABLE 2 Effects of Phosphate Substrate on Cell Growth Strain JM101 JM101JM101 GB993 GB993 GB993 0.1 mM 5 mM 0.2 mM 0.1 mM 5 mM 0.2 mM PhosphatePhosphate AMPA Phosphate Phosphate AMPA Growth Period (hrs) 48 29 54 4829 54 Harvest Density 155 240 — 140 244 185 (Klett Units) — indicates nomeasurable growth

An HPLC assay was used to determine the presence or absence of any AMPAacyltransferase activity in the media and cell lysates. The assaymonitors the conversion of [¹⁴C]AMPA to N-acetyl-[¹⁴C] AMPA. Generally,100 μl of a 2× assay solution consisting of 16.5 mg acetyl-CoA, 250 μlof 2M Tris, pH 7.5, 4.5 mls dH₂0 and [¹⁴C]AMPA (30 mM) was mixed with25-75 μl of lysate and 1 μl each of 0.5 M MgCl₂ and MnCl₂, and broughtto 200 μl with dH₂0. The assay was incubated for 30 minutes at 37° C.,and quenched with 200 μl 90-100 mM NaOAc (sodium acetate) pH 4.4 inethanol and then analyzed immediately by HPLC as described above, orstored at −20° C. Only GB993 lysate samples derived from cultures grownin the presence of AMPA or 0.1 mM phosphate supplemented mediademonstrated appreciable AMPA acyltransferase activity. This resultindicated that a gene encoding an acyltransferase enzyme capable of AMPAN-acylation was present in GB993 and was regulated for expression whengrown under low phosphate conditions. Thus, the coding sequence for theenzymatic activity appears to be part of the pho regulon and may residein the phn operon.

Example 4

This example illustrates the identification of an E. coli phn operongene encoding an enzyme capable of AMPA acylation.

Example 3 indicated that the AMPA acyltransferase activity observed inlysates of E. coli may be encoded by a gene in the phn operon. Theentire phn operon in E. coli B and in E. coli K-12 has previously beencloned and sequenced B (Wanner et al., Chen et al.). The E. coli K-12phn operon DNA sequence has been shown to be identical to the publishedDNA sequence of the phn operon from E. coli B with the exception of aneight base pair insertion in the phnE gene (Wanner et al). Clonescontaining various amounts of the phn operon genes from either bacterialgenetic background are readily available (Wanner et al., Chen et al.,Dr. J. W. Frost at Purdue University). Plasmids containing differingamounts of the JM101 phn operon DNA were used to transform JM101 (mpu−)in order to test for a plasmid localized phn gene that, when expressed,confers upon JM101 the ability to utilize AMPA as a sole phosphatesource.

A plasmid obtained from J. Frost (Dr. J. W. Frost, Department ofChemistry, Purdue University, West Lafayette, Ind. 47907), designatedherein as pF, contains an E. coli K-12 8 kb EcoRI fragment which encodesthe phn operon genes phnG through phnQ. A single NcoI site is present atthe 5′ end of the phnG coding region. Plasmid pF was digested with EcoRIand NcoI, releasing a 2 kb NcoI-EcoRI fragment containing the genes phnGthrough phnI, and a second NcoI-EcoRI fragment about 6 kb in lengthcontaining the genes phnJ through phnQ. Each fragment was gel purifiedand ligated into a cloning and expression vector in an orientation whichwould allow for expression of the phn operon genes present within eachof the NcoI-EcoRI fragments from a plasmid borne inducible promoter. The2 kb fragment was inserted into the NcoI-EcoRI sites within the vectorpMON7258, a positive selection cloning vector identical to pUC118 withthe exception of polylinker domain (Viera et al., Methods Enzymol.153:3, 1987), the resulting plasmid being designated as p58-1. Theorientation of the 2 kb fragment in p58-1 allows for the expression ofthe phnG-phnI genes from the lac promoter within the vector. The 6 kbEcoRI-NcoI fragment was inserted into the NcoI and EcoRI sites in asimilar positive selection vector, pMON7259, producing the plasmiddesignated as pMON17195. pMON7259 is identical to pUC119 except for thepolylinker domain, which contains a multiple cloning site opposite inorientation to that within pMON7258, and which also allows forexpression of the phnJ-phnQ genes from a lac promoter. p58-1 andpMON7259 were transformed into E. coli K12 (mpu−) strain JM101, andmaintained with ampicillin antibiotic resistance selection. pMON7259 andpF were also transformed into JM101 as negative and positive controls,respectively.

Cultures of each transformant were grown overnight in M9 liquid brothmedia supplemented with 2% casamino acids, thiamine, and 0.2% glucosewith shaking at 37° C., and then diluted 1:50 into 50 ml of freshpre-warmed media of the same composition in a 250 ml side-armedErlenmeyer flask. Cultures were incubated with shaking at 37° C. untilreaching a cell density of about 80-100 Klett Units as measured on aKlett-Summerson spectrophotometer through a #2 green filter. Expressionfrom the plasmid lac promoter was induced by the addition of 100microliters of 500 mM IPTG so that the final IPTG concentration wasabout 1 mM. The induction phase growth period was allowed to progressfor two hours. Table 3 shows the cell density profile of each culturefrom 1:50 dilution through the two hour induction period.

TABLE 3 Induction Profile of JM101 Cultures Harboring Various phnPlasmids Plasmid Culture IPTG I₀ I₁ I₂ pMON7259 + 13 75 222 p58-1 + 1570 212 pMON17195 + 15 90 220 pF + 17 97 290 pF − 15 — 260 I₀ indicatesthe cell culture density at the 1:50 dilution time point; I₁ indicatesthe cell culture density at the time of IPTG addition; and I₂ indicatesthe cell culture density at the time of harvest.

The cells in each culture were harvested by centrifugation at 10,000 rpmfor 10 minutes at 4° C. in a Beckman J2 centrifuge. The cell pellet waswashed one time in ice cold 154 mM NaCl solution, and then resuspendedin 1.5 ml extraction buffer (50 mM Tris-HCl pH 7.5, 1 mM DTT, 50 mMTris-HCl pH 7.5). Cell suspensions were ruptured with two passes througha French Press at 1000 psi. The resulting lysate was centrifuged for 15minutes at 14,000 rpm at 4° C. in an EPPENDORF™ model 5402microcentrifuge in order to remove debris. Each cleared lysate wastransferred to a fresh pre-chilled tube and the volume of the extractwas adjusted to 2.5 ml with 50 mM Tris-HCl pH 7.5. A PD10 column wasequilibrated with 25 ml 50 mM Tris-HCl, pH 7.5 and then each sample wasapplied to the desalting column. Each eluted sample was adjusted to 3.5ml with 50 mM Tris-HCl, pH 7.5. Each sample was distributed to assaytubes and mixed with reagents in order to assay for the presence of AMPAacyltransferase activity as shown in Table 4.

TABLE 4 Assay Conditions for Bacterial Lysates Expressing phn Genes 2XAssay Extract 50 mM Tris Mix Total Sample IPTG Volume* Volume* Volume*Volume* pMON7259 + 25 75 100 200 pMON7259 + 100 0 100 200 p58-1 + 25 75100 200 p58-1 + 100 0 100 200 pMON17195 + 25 75 100 200 pMON17195 + 1000 100 200 pF + 25 75 100 200 pF + 100 0 100 200 pF − 25 75 100 200 pF −100 0 100 200 — na 0 100 100 200 *all volumes are in microlitersComposition of mixtures of each sample, designated by plasmid content,as prepared for AMPA acyltransferase assay.Each mixture was incubated at 37° C. for 30 minutes, and quenched withan equal volume (200 microliters) of 90-100 mM NaOAc (sodium acetate),pH 4.4 in ethanol and if not analyzed immediately by HPLC as describedabove, then stored overnight at −20° C. Unused portions of each lysatewere stored either at 4° C., or mixed with glycerol to 10% by volume,and stored at −20° C.

Samples of each lysate subjected to the AMPA transacylase assay wereanalyzed by HPLC for the presence of [¹⁴C]AMPA and acylated [¹⁴C]AMPA,as described above. The results are shown in Table 5.

TABLE 5 HPLC Analysis of Bacterial Lysate Conversion of AMPA toAcetyl-AMPA Sample % Acetyl AMPA % AMPA pMON7259 no data no datapMON7259 8 92 p58-1 5 95 p58-1 13 87 pMON17195 100 0 pMON17195 100 0 pF61 39 pF 97 3 pF 52 48 pF 90 10 — — 100 Results of HPLC analysis of eachsample, indicating the relative amount of [¹⁴C] AMPA or acetyl-[¹⁴C]AMPAas a percentage of the total amount of [¹⁴C] in both peaks combined.

This data indicated that the plasmid containing the 6 kb NcoI-EcoRIfragment isolated from pF in pMON17195 contained one or more geneswhich, upon IPTG induction of the lac promoter in an mpu− strain of E.coli, elicited the production of an acyltransferase activity capable ofconverting all of the [¹⁴C]AMPA available in the assay mix toacetyl-[¹⁴C]AMPA. The gene or genes required for AMPA N-acylation werefurther defined by restriction deletion analysis.

Plasmids containing various segments of the phn operon from either E.coli B or E. coli K-12 were constructed to further delineate the natureof the phn operon gene or genes involved in conferring AMPAacyltransferase activity when expressed in an mpu− E. coli JM101.pMON7333 contains the pMON17195 equivalent E. coli DNA insertion, but inpUC119, and is a single E. coli B strain HindIII fragment containing thewild type phn operon genes phnG through phnQ. pMON15020 was constructedby cloning a 5,713 base pair NcoI to EcoRI E. coli B DNA fragment frompMON7333 into pMON7259, and contains the genes phnJ through phnQ.pMON15022 was constructed by inserting a 1,686 base pair EcoRI to SalIfragment from pMON17195 into the positive selection cloning andexpression vector pBlueScriptSP (Invitrogen), which contains the E. coliK-12 genes phnO, P and Q. pMON15023 was constructed by deleting an 1,820base pair SalI fragment from pMON17195, leaving behind the E. coli K-12phn operon genes phnJ and phnK, the 5′ end of phnL, and all of phnO, Pand Q.

The plasmids pMON17195, pMON15020, pMON15022, pMON15023, and pMON7259were transformed into the mpu− E. coli K-12 strain JM101 and weremaintained by ampicillin antibiotic selection. Overnight cultures ofeach of these transformants were grown with antibiotic selection andwere diluted 1:50 into fresh M9 media as described above, and incubatedat 37° C. with shaking in 250 ml sidearm-Erlenmeyer flasks to a celldensity of about 100 Klett units. Each culture was induced with IPTG asin example 3, and incubated for two additional hours with shaking. Thecells were harvested by centrifugation in a Beckman J2 centrifuge at4,000 RPM for 10 minutes at 4° C. Cell pellets were washed once with 50ml of 154 mM NaCl, and stored at −20° C.

Cell pellets were resuspended in 1.5 ml Extraction Buffer as in example3 and ruptured by two passes through a French Press at 1000 psi. Theruptured cell suspensions were centrifuged in an Eppindorfmicrocentrifuge Model 5402 for 15 minutes at 14,000 rpm and at 4° C. Thecleared lysates were decanted into new tubes pre-chilled on ice, and thetotal volume was adjusted to 2.5 ml with addition of Extraction Buffer.These samples were desalted over a PD10 column pre-equilibrated with 25ml of 50 mM Tris-HCl, pH 7.5, and eluted with 3.5 ml of 50 mM Tris HClpH 7.5. Samples were then subjected to an AMPA acylation assay asdescribed above, incubated for 30 minutes at 37° C., and quenched with200 microliters of 90.9 mM NaOAc pH 4.4. The volumes of each sample usedin the assay are noted in Table 6. All volumes represent microliters ofeach solution used.

TABLE 6 Assay Conditions for Bacterial Lysates Expressing phn Genes fromPlasmids Plasmid Extract 50 mM Tris 2X Assay Mix Total Volume — — 100100 200 pMON 17195 25 75 100 200 pMON 17195 100 — 100 200 pMON 15020 7575 100 200 pMON 15020 100 — 100 200 pMON 15022 75 75 100 200 pMON 15022100 — 100 200 pMON 15023 75 75 100 200 pMON 15023 100 — 100 200 pMON7259 75 75 100 200 pMON 7259 100 — 100 200 Composition of mixtures ofeach sample, designated by plasmid content, as prepared for AMPAacyltransferase assay

Quenched samples were subjected to HPLC analysis as described above.Table 7 illustrates the results of HPLC analysis of each sample,indicating the relative amount of [¹⁴C] AMPA or acetyl-[¹⁴C]AMPA as apercentage of the total amount of [¹⁴C] in both peaks combined.

TABLE 7 HPLC Analysis of Bacterial Lysate [¹⁴C]-AMPA Conversion toAcetyl-[¹⁴C]-AMPA Extract %[¹⁴C]- Total % Sample Volume AMPA %Acetyl-[¹⁴C]-AMPA [¹⁴C] — — 100 — 100 pMON17195 25 66 34 100 pMON17195100 26 74 100 pMON15020 75 — 100 100 pMON15020 100 — 100 100 pMON1502275 — 100 100 pMON15022 100 — 100 100 pMON15023 75 — 100 100 pMON15023100 — 100 100 pMON 7259 75 87 13 100 pMON 7259 100 72 28 100 HPLCanalysis of each sample, indicating the relative amount of [¹⁴C] AMPA oracetyl-[¹⁴C]AMPA as a percentage of the total amount of [¹⁴C] in bothpeaks combinedThe data in Table 7 indicates that AMPA acylation activity is derivedfrom the phn operon open reading frames consisting of phnO, phnP, andphnQ, which are the only phn genes present in pMON15022. Other plasmidsconferring AMPA acylation activity upon induction also contained atleast the phnO, P, and Q genes, providing strong evidence that theobserved activity was the result of one or more of these gene products.Therefore, additional plasmids were constructed based on the phnO, P,and Q gene sequences in order to determine which gene or genes wererequired for the acylation function.

Bacterial acylase, transacylase, and acyltransferase genes have beenknown in the literature for some time. Most are small 15-25 K Daproteins. Therefore, on the basis of size comparison, only the phnO andphnQ gene products would fall into this category. However, based onsimilarity comparisons with other proteins in the GENBANK, SWISSPROT,and EMBL databases, the predicted phnO gene product appeared to mostclosely resemble other proteins having acylase activity. For example,the E. coli PhnO protein aligned well with a gentamicinacetyltransferase-3-I described in Wohlleben et al. (Mol. Gen. Genet.217:202-208, 1989). pMON15020 containing the E. coli B phn operon genesphnJ through phnP on a single 6.0 kb NcoI-EcoRI fragment was digestedwith SalI and EcoRI to release a 2.0 kb fragment containing the phnO, Pand Q genes. This 2 kb fragment was excised and purified from a 0.7% TAEAgarose gel, treated with T4 DNA polymerase to excise the 3′ overhangingends, then with Klenow and deoxynucleotide triphosphates (dXTP's) toprovide blunt ends, and then ligated into the EcoRV site ofpBlueScriptSP to produce plasmid pMON15024. pMON15024 was digested withNdeI and EcoRI, deleting a 1200 base pair fragment containing most ofthe phnP and all of the phnQ coding sequences. The remaining pMON15024plasmid fragment still containing the phnO gene was treated with Klenowfragment DNA polymerase in the presence of dideoxynucleotides accordingto the manufacturer's instructions in order to fill in the 3′ endsexposed by restriction enzyme digestion, then ligated together toproduce the plasmid pMON15027. pMON15027 contains only the phnO geneflanked 3′ by a small portion of phnP. The 1200 base pair NdeI to EcoRIfragment obtained from pMON15024 was cloned into pMON2123 to producepMON15026, which contains the 3′ two thirds of the phnP gene flanked 3′by phnQ. Plasmids pMON15024, 15026, and 15027 were introduced into mpu−JM101, and cell lysates of transformants were analyzed as above aftergrowth and induction for the presence of AMPA acyltransferase activity.Only pMON15024 and pMON15027 exhibited acyltransferase activity,indicating that the phnO gene product was responsible for AMPAacylation.

A DNA fragment containing only the phnO gene with convenient flankingrestriction endonuclease sites for use in further cloning manipulationswas produced using thermal cycling methods. Synthetic oligonucleotideprimers were synthesized by Midland Certified Reagents, Co. (MidlandTex.) based on the published phnO gene and flanking sequence in order toamplify the phnO gene (Chen et al., J. Biol. Chem. 256: 4461-4471,1990). The sequence AAACACCATGGCTGCTTGTG (SEQ ID NO: 5), designatedAATPCR6, represents a synthetic oligonucleotide which is homologous tothe template strand of the phnO gene. The 5′ adenosine residue of SEQ IDNO: 5 corresponds to base pair 13,955 of the published phn operonsequence, immediately 5′ of the phnO ATG initiation codon at position13,962-13,964 (Chen et al., J. Biol. Chem. 256: 4461-4471, 1990). SEQ IDNO: 5 incorporates a single base pair mismatch from the published phnOsequence at position 13,965 represented by a C to G inversion, whichgenerates an alanine codon in place of a proline codon at position 2 andalso creates a unique NcoI restriction site spanning the ATG initiationcodon. The sequence GTGACGAATTCGAGCTCATTACAGCGCCTTGGTGA (SEQ ID NO: 6),designated AATPCR7, represents a synthetic oligonucleotide which ishomologous to the coding strand of the phnO gene. The 3′ adenosineresidue of SEQ ID NO: 6 corresponds to base pair 14,380 of the publishedphn operon (Chen et al., J. Biol. Chem. 256: 4461-4471, 1990). Thethymidine at position number nineteen of SEQ ID NO: 6 corresponds to theadenosine at position 14,396 of the published phnO sequence (Chen etal.). A portion of SEQ ID NO: 6 overlaps the native phnO terminationcodon, introduces a second in frame termination codon immediately 3′ ofand adjacent to the native termination codon, and also introduces uniqueEcoRI and SacI restriction sites 3′ of these termination codons.

pMON15024 was used as a template for amplification of the phnO gene in astandard thermal amplification reaction. Briefly, a 100 microliterreaction sample was prepared which contained 0.1 ng template DNA,reaction buffer, 200 pM each primer, 200 mM dNTP, 1.25 U Taq DNApolymerase and was overlayed with mineral oil. This reaction sample wassubjected to thirty five cycles at 94° C. for one minute, 50° C. for twominutes, and 72° C. for three minutes which resulted in theamplification of a 459 base pair DNA product as determined by analysisof five microliters of the reaction sample on a ethidium bromide stained0.7% TAE agarose gel. A 444 base pair product was purified usingstandard methods from a 1% TAE agarose gel after digestion of a sampleof the 459 base pair amplification product with NcoI and EcoRIrestriction endonucleases. The 444 base pair product was ligated intocompatible sites in pMON7259 to generate pMON15028. Cell lysatesprepared as above from IPTG induced cultures of JM101 containingpMON15028 were analyzed for the presence of AMPA acyltransferaseactivity and compared to cultures containing pMON15027. The results wereindistinguishable, thus confirming that phnO encoded an enzyme capableof AMPA acylation. In addition, this result indicated that the P2Amutation in the protein, which was introduced into the gene codingsequence as a result of thermal amplification using the AATPCR6oligonucleotide primer (SEQ ID NO: 5), was without effect on theacyltransferase activity of the resulting PhnO protein when expressed inE. coli.

Example 5

This example illustrates the production of polyclonal antibodiesdirected to the PhnO peptide.

Further studies of the phnO gene product required the use of antibodiesdirected to the PhnO protein. Therefore, PhnO was overproduced in E.coli JM101 for use as an immunogen in stimulating the production ofantibodies upon injection into a goat. The phnO gene containing the P2Amutation in plasmid pMON15028 was introduced into plasmid pMON17061 onan NcoI to EcoRI DNA fragment, producing pMON15032. phnO expression inpMON15032 is under the control of the E. coli recA promoter adjacent tothe bacteriophage T7 gene 10 L ribosome binding sequence. Cells weregrown to mid log phase and induced by addition of nalidixic acid to theculture to approximately 50 parts per million, from a stock solution of50 mg nalidixic acid powder dissolved in 1 ml 0.1 N NaOH. The culturewas maintained under inducing conditions for twelve hours at 37° C.Cells were harvested as described in example 3, and sonicated inphosphate buffered saline. About 23% of the total soluble protein in theinduced E. coli lysates was determined to be PhnO and approximately 60%of the total PhnO protein was released into the soluble phase as judgedby SDS-PAGE and Coomassie blue staining. The protein was furtherpurified by preparative SDS-PAGE providing a sufficient quantity of PhnOfor use in producing antibody which binds to or reacts antigenicallywith PhnO or related AMPA transacylase proteins. Briefly, the PhnOprotein was separated by size from other proteins in a 15% SDS-PAGE gel.A gel slice containing the PhnO protein was excised, weighed, andhomogenized using a polytron in a volume of phosphate buffered saline(PBS, pH 7.0) equal to the mass of the gel slice. The homogenate wasmixed with an equal volume of complete Freund's media until a colloidalmixture was obtained. An 8-ml inoculum of this mixture was used for thefirst injection into a goat. Two weeks post-injection, a 50-ml bleed wascollected and serum was separated from blood solids by centrifugation. Abooster injection of gel purified PhnO protein was administered in acolloidal mixture of 50% incomplete Freund's adjuvant at four weeks, andat six weeks a second bleed was obtained.

The serum from the second bleed was used to screen for the presence ofsufficient antibody titers specific for PhnO protein. Extracts fromJM101 cells containing pMON15032 were subjected to western blotanalysis. The concentration of protein in the extract was determined tobe about 55 mg/ml by Bradford assay, and a prior Coomassie stained gelusing this same extract was subjected to a densitometer scan whichindicated that about 23% of the total cell protein was PhnO. The extractwas desalted over a PD10 column, eluted with 10 mM Tris pH 7.5, anddiluted with an equal volume of 2×SDS sample buffer. Serial dilutionswere prepared using 1× sample buffer and loaded into wells of a 15% SDSPAGE gel. Additional samples were mixed with a tobacco leaf proteinextract containing 10 additional micrograms of protein per lane inaddition to the E. coli PhnO extracts. The tobacco leaf protein extractswere used to screen for the presence of cross reactive antibody to plantproteins. Proteins were separated according to size by electrophoresisat 7.5 mA constant for fourteen hours at 4° C., and the gel waselectroblotted onto a MSI 0.45 micron nitrocellulose filter at 0.5Ampere in Tris-Glycine transfer buffer for one hour. The membrane wasthen blocked with TBST (Tris, BSA, NaCl, Tween-20, Short Protocols inMolecular Biology, 3rd Ed., Wiley and Sons, Pub.) for two hours at roomtemperature, incubated forty-five minutes with a 1:500 dilution of thesecond bleed serum at room temperature, washed two times in TBST,incubated another forty-five minutes with alkaline phosphataseconjugated rabbit anti-goat IgG (Boehringer Mannheim Biochemicals,Inc.), washed three times with TBST and one time with alkalinephosphatase buffer, and finally incubated for two and one half minuteswith a standard color development solution containing NBT and BCIP. Thereaction was terminated by washing the membrane with ample quantities ofdistilled water. The antibody was able to detect PhnO protein in aslittle as 50 nanograms of E. coli extract independent of the presence ofadditional plant proteins in one half of the samples. In addition, veryfew cross reactive bands were detected in either set of samples,indicating that the serum sample contains very little IgG which crossreacts with either E. coli or tobacco plant proteins when tested usingthis western blot method.

An alternative source for generating antibody which is capable ofspecific binding to or reacting antigenically with PhnO protein was alsoutilized. A phnO gene was placed into a commercial vector (Invitrogen)containing a metal binding amino acid coding sequence (His6) upstream ofand in frame with the phnO coding sequence. The His 6-phnO DNA sequencewas inserted into the E. coli expression vector pMON6235 on an NcoI toEcoRI fragment, under the control of an E. coli arabinose operon araBADpromoter, producing plasmid pMON32909. His6-PhnO protein was producedupon arabinose induction of E. coli W3110 cells containing pMON32909,and purified over a metal affinity column according to themanufacturers' instructions.

His-tagged purified His6-PhnO protein standard was injected into 6 NewZealand White rabbits using an immunization procedure similar to thatused for the goat, described above. Antiserum raised in these rabbitswas also shown to be specific for binding PhnO protein and non-crossreactive with other E. coli bacterial or tobacco plant proteins.

Example 6

This example illustrates properties of an AMPA transacylase enzyme usingaminomethylphosphonate and acetyl-CoA as substrates in an enzyme assayas measured by endpoint kinetic analysis.

The apparent Km (Km) and Vmax (Vmax) of PhnO enzyme were determined forthe substrates aminomethlyphosphonate and acetyl-CoA. Determination ofthe PhnO Km and Vmax were made by endpoint kinetic analyses, determiningthe enzyme velocity in consuming each substrate at varying substrateconcentrations, and plotting the inverse of the enzyme velocity versusthe inverse of the substrate concentration to produce a Lineweaver-Burkplot of enzyme kinetics. The conversion of [¹⁴C]-AMPA toN-acetyl-[¹⁴C]-AMPA was monitored as in example 2, using enzyme in adesalted crude lysate of E. coli expressing phnO from pMON15032,produced as in example 4. Total protein per ml of extract was determinedby the method of Bradford which indicated approximately 22.5 mg/ml.Densitometric scanning of Coomassie stained SDS-polyacrylamide gelsresolving PhnO protein from these lysates indicated that PhnO representsabout 23% of total protein, thus the cell extract was determined tocontain about 5.2 mg PhnO protein per ml. In a first assay to determinethe apparent Km and Vmax of PhnO for AMPA, [¹⁴C]-AMPA concentrationsranged from 2 to 38 mM. Enzyme reactions were incubated at 37° C. for 5minutes and quenched with 1 volume of 100 mM sodium acetate (NaOAc), pH4.4, in ethanol. Samples were analyzed by HPLC to determine the amountof [¹⁴C]-AMPA converted to N-acetyl-[¹⁴C]-AMPA. The assay conditions andoutput for each set of reactions are shown in Table 8.

TABLE 8 PhnO Enzyme Kinetics for AMPA Substrate Sample S¹ % Turnover²Velocity³ 1/S 1/V V/S 1 200 39.5 79 1.0 0.0127 79.00 2 400 35.1 140 0.50.0071 70.00 3 800 32.9 263 0.25 0.0038 65.75 4 1200 26.8 322 0.1660.0031 53.67 5 1600 26.2 426 0.125 0.0023 53.25 6 2000 22.1 442 0.1000.0023 44.20 7 2400 19.2 461 0.083 0.0022 38.42 8 2800 17.6 493 0.0710.0020 35.21 9 3200 17.3 554 0.063 0.0018 34.63 10 3600 14.5 522 0.0560.0019 29.00 11 4000 13.6 544 0.050 0.0018 27.20 12 6000 12.7 762 0.0330.0013 25.15 13 7600 10 760 0.026 0.0013 19.76 ¹AMPA substrateconcentration in reaction in nm (nanomoles) ²% turnover measured by thepercent of N-acetyl-[¹⁴C]-AMPA formed in relation to the amount of[¹⁴C]-AMPA remaining in the sample ³enzyme velocity in units of AMPA(nm) converted to N-acetyl-AMPA per minute per mg of proteinA Linweaver-Burk plot of the 1/V vs. 1/S data from Table 8 indicatesthat the apparent Km of PhnO for AMPA as a substrate is about 9 mM, andthe apparent Vmax is about 824 U/mg protein.

The apparent Km of PhnO for the substrate acetyl-CoA was determined insimilar experiments. After several attempts to obtain end pointkinetics, it was determined that the turnover number was too low to bereliable at AMPA concentrations of about 30 mM and enzyme amounts ofabout 1-10 ng. An alternative approach was tried using tritium labeledacetyl-CoA. The specific activity of the label was about 40× higher thanwith [¹⁴C], providing a gain in sensitivity that allowed for thedetermination of the apparent Km of PhnO for Acetyl-CoA. The[³H]-acetyl-CoA (Amersham, Inc.) specific activity was 360 mCi/mg or 250μCi/ml. The transacylation mediated by PhnO from [³H]-acetyl-CoA to[³H]-acetyl-AMPA was monitored by weak anion exchange HPLCchromatography, with the retention times of acetyl-CoA and acetyl-AMPAadjusted so that these compounds were separated by about three minutes.This was accomplished by adjusting the concentration of KH₂PO₄ buffer(pH 5.5) to 40 mM with a flow rate of 1 ml per minute over an AX100 weakanion exchange column. Each sample was reacted with PhnO and 30 mM AMPAfor five minutes at 37° C. and quenched with 100 mM NaOAc pH 4.4 inethanol, then analyzed by HPLC. [³H]-acetyl-CoA substrate ranged from 25micromolar to 1.3 mM in each reaction along with about 5 ng PhnO, 50 mMTris pH 7.5, 1 mM MnCl₂, 1 mM MgCl₂, and 30 mM AMPA. Samples wereanalyzed by HPLC to determine the amounts of N—[³H]-acetyl-AMPAproduced, and [³H]-acetyl-CoA remaining. The assay conditions andresults for these reactions are shown in Table 9.

TABLE 9 PhnO Enzyme Kinetics for Acetyl-CoA Donor Substrate Sample No.[Acetyl-CoA]¹ Velocity² 1/[S]³ 1/V⁴ V/S⁵ 1 25 34 0.0400 0.0294 1.3600 250 66 0.0200 0.0152 1.3200 3 75 94 0.0133 0.0106 1.2533 4 100 125 0.01000.0080 1.2500 5 125 150 0.0080 0.0067 1.2000 6 150 173 0.0066 0.00581.1533 7 175 193 0.0057 0.0052 1.1029 8 200 219 0.0050 0.0046 1.0950 9225 240 0.0044 0.0042 1.0667 10 250 259 0.0040 0.0039 1.0360 11 375 3390.0027 0.0030 0.9040 12 390 287 0.0026 0.0035 0.7359 13 520 331 0.00190.0030 0.6365 14 650 352 0.0015 0.0028 0.5415 15 780 372 0.0013 0.00270.4769 16 910 397 0.0011 0.0025 0.4363 17 1040 411 0.0009 0.0024 0.395218 1170 425 0.0008 0.0024 0.3632 19 1300 434 0.0007 0.0023 0.3338¹substrate concentration in micromolar units ²enzyme velocity asmeasured by amount of [³H] incorporated into [³H]-acetyl-AMPA per unittime ³inverse substrate concentration ⁴inverse velocity ⁵ratio ofvelocity to substrate concentrationA Linweaver-Burk plot of the 1/V vs. 1/S data from Table 9 indicatesthat the apparent Km of PhnO for acetyl-CoA as a substrate is between375-390 micromolar, and the apparent Vmax is about 824 U/mg protein.

An approximate pH range of activity for the PhnO enzyme was determinedusing enzyme in a crude lysate of E. coli expressing phnO frompMON15032. The ability of the enzyme to produce N-acetyl AMPA from amixture containing acetyl-CoA and AMPA across a range of pH values wasdetermined. The reactions were carried out in MES/MOPS/Tricine bufferequilibrated to a pH value from 4.5 to 9.0, with actual pH valuesranging from 5.2 through 9.0. Briefly, 95 microliters of an appropriatebuffer was mixed with 100 microliters of 2× assay mix as described inexample 4, and 5 microliters of desalted E. coli lysate containingapproximately 400 ng/microliter PhnO protein. The reaction was incubatedat 37° C. for five minutes and quenched with 100 mM NaOAc pH 4.4 inethanol, and analyzed by HPLC as described in example 4. The results areshown in Table 10.

TABLE 10 PhnO Enzyme pH Profile ³Velocity Buffer ¹Mock ²% N-Acetyl CoA(nmole/min/ pH Reaction pH Turnover (nmole) microgram) 5.0 5.23 3.7 22222.2 5.5 5.62 3.9 234 23.4 6.0 5.92 4.2 252 25.2 6.5 6.47 13.3 798 79.87.0 7.0 27.0 1620 162.0 7.5 7.48 32.0 1920 192.0 8.0 8.05 34.3 2058205.8 8.5 8.46 33.5 2010 201.0 9.0 9.0 33.9 2034 203.4 ¹indicates truepH value after combining all reagents for each initial buffer pH valuegiven ²determined as in Table 9 for Km and Vmax ³determined as in Table9 for VmaxThe results indicate that optimum PhnO transacylase activity using AMPAand acetyl-CoA as substrates is about pH 8.0. However PhnO efficientlyconverts AMPA to N-acetyl-AMPA using acetyl-CoA as the acetyl donoracross a pH range from about 6.5 to at least 9.0.

Additional experiments were carried out with purified PhnO protein tofurther characterize the scope of the enzyme's substrate preference foracyl-CoA acyl donor compounds. It has been established herein that atleast one substrate acyl-donor or leaving group can be a two carbon acidcompound such as the acetyl-moiety in the compound Acetyl-CoA. It wasnot known what range of acyl-molecules comprised of different carbonchain lengths would or could function as a leaving group from theacyl-CoA acyl donor when reacted with PhnO transacylase and AMPA as theacyl-receptor molecule. Therefor, an HPLC assay similar to thatdescribed in Example 2 was developed to determine the scope of theenzymes' ability to transfer an acyl-group from an acyl-CoA acyl donorto [¹⁴C]-AMPA.

PhnO was purified from a one liter Luria Bertani broth culture of E.coli JM101 expressing a recombinant phnO gene from pMON15032 afternalidixic acid induction for three hours at 37° C. Cells were harvestedby centrifugation and resuspended in 40 ml cold Tris buffer (0.1 MTris-HCl pH 8) and placed on ice. The cell suspension was brought to 1mM DTT and 0.5 mM PMSF. The suspension was lysed by 2 passages through aprechilled French pressure cell at 1,100 psi, centrifuged at 12,000 g(10,000 rpm in an Sorvall SA600 rotor) for 40 min at 4° C., then placedon ice. The cleared supernatants were poured into fresh 15 mlpolypropylene tubes. The samples were split again into two equalportions and maintained at −80° C. until used further for purificationof PhnO protein. 20 microliters of the soluble fraction was assayed forenzyme activity using the HPLC method described above in Example 2,except after terminating the assay with acid addition, the sample wasstored at −80° C. A Sephacryl S200 column was prepared according to themanufacturers' instructions and equilibrated with a solution containing20 mM Tris pH 8.0 and 0.5 mM MgCl₂. The entire total soluble extract waslayered over the top of the column bed after thawing on ice. Forty 9 mlfractions were collected from the column eluate, and thirty microlitersof each fraction was analyzed by western blot using anti-PhnO antiserumafter resolution on a 15% SDS-PAGE gel. Also, thirty microliters of eachfraction was analyzed for AMPA acyl transferase activity using themethod described in Example 2. Samples which exhibited acyl transferaseactivity and which corresponded to positive western blot data werepooled. These were represented by fractions 7 through 19 in thisexample, and were combined into a 100 ml volume, distributed into ten 10tubes each containing 10 ml volumes, and stored at −80° C. for furtheruse.

Anion exchange chromatography was used to determine the elution patternof PhnO away from other contaminating proteins that co-elute during theSephacryl S200 fractionation. One tube from the combined PhnO positivefractions was thawed on ice and injected into a 5/5 Mono-Q columnpre-equilibrated with buffers A (one liter of 20 mM Tris-HCl pH 8.0Mili-Q distilled deionized water) and B (one liter of 20 mM Tris-HCl pH8.0, 1 M NaCl). The sample containing PhnO active protein was injectedinto the column and one milliliter fractions were collected. The columnwas washed for five minutes with a flow rate of 1.8 ml per minute BufferA after loading the PhnO containing sample. At five minutes, Buffer Bwas added to the flow volume at 0.5 ml per minute for four minutes.Buffer B was ramped up to 22% of the flow volume at 10 minutes, 30% at12 minutes, 36% at 13 minutes, 41% at 14 minutes, 46% at 15 minutes, 74%at 16 minutes, and 100% at 16 minutes through 22 minutes, at which pointBuffer B flow was terminated and Buffer A was reinitiated at 100% toequilibrate the column. Ten microliter volumes from individual fractionscollected from the Mono-Q column were analyzed by western blot and fortransacylase activity as described in Example 2. Fractions whichexhibited positive AMPA acyltransferase activity and which correlatedwith the Western blot data were pooled and maintained as a purifiedprotein sample. Samples of this purified PhnO protein were used todetermine enzyme's acyl donor substrate specificity.

Enzyme reactions were prepared as follows. 100 microliter reactionsconsisted of 50 mM Tris-HCl pH 8.0, 1 mM MgCl₂, 3 microliters of 1.3 mM[¹⁴C]-AMPA (115,392 dpm per microliter), 0.1 mM or 1 mM acyl-CoA acyldonor, and 2.5 microliter purified enzyme sample. A assay premix wasprepared from which 45 microliters was used in each 100 microliterreaction. This 45 microliter premix sample consisted of 40 microlitersdistilled and deionized water, 2 microliters of 50 mM MgCl₂, and 3microliters of 1.3 mM [¹⁴C]-AMPA (115,392 dpm per microliter). Reactionswere initiated by mixing 40 microliters of 125 mM Tris-HCl pH 8.0, 2.5microliters protein sample and 10 microliters acyl-CoA acyl donorcompound in a microcentrifuge tube at room temperature. Each acyl-CoAacyl donor compound was prepared as a stock solution of 1 mM, 5 mM or 10mM stocks. Each tube was then mixed with 45 microliters of the assaypremix containing the [¹⁴C]-AMPA receptor substrate, mixed gently andtransferred to a 30° C. water bath for 5 minutes. Each reaction wasterminated with the addition of 4 microliters of 1M HCl, mixed byvortexing, and placed on ice or stored at −20° C. until assayed for thepresence of [¹⁴C]-AMPA or related compounds by HPLC.

HPLC analysis was carried out using a Waters 510 dual pump HPLC systemwith a 481 wavelength max UV detector and a scintillation pump, aPhenomenex PHENOSPHERE 5 micrometer 80 Å SAX-silica HPLC column (250×4.6mm, 3500 PSI max pressure), Buffer A consisting of 5 mM KH₂PO₄, 4%methanol, adjusted to pH 2.0 with H₃PO₄, and Buffer B consisting of 200mM KH₂PO₄, 4% methanol adjusted to pH 2.0 with H₃PO₄, and HAZARDAtomflow (Packard) containing 64% 1,2,4 trimethylbenzene, 7.5%sodium-dicotyl sulfosuccinate, 3.5% sodium diamylsulfosuccinate, and 6%polyoxyethylene(4)lauryl ether. HPLC gradient conditions for each sampleanalysis were similar to those described in Example 2, with minorvariations. The flow rates are provided in Table 11.

TABLE 11 HPLC Gradient Conditions Time Flow (min) (ml/min) % A % B FlowRate¹ 0.0 1 100 0 3 2.0 1 100 0 3 5.0 1 50 50 3 15.0 1 0 100 3 17.0 1 0100 3 17.3 1 100 0 3 21.0 1 100 0 3 21.3 0.1 100 0 0 ¹Scintillationfluid flow rate in milliliters per minute

Stock solutions of Acyl-CoA acyl donor compounds were prepared asdescribed above, and these are listed here: Na Acetyl-CoA, Lin-propionyl-CoA, Li glutaryl-CoA, Li methylmalonyl CoA, Licrotonoyl-CoA, Li isobutyryl-CoA, Na succinyl-CoA, Li tiglyl-CoA, Lin-valeryl-CoA, and Li desulfo-CoA. All compounds were obtained fromSigma Chemical Company, St. Louis, Mo. The percent activity of thepurified enzyme for transfer of the CoA associated acyl-moiety to[¹⁴C]-AMPA was determined by measuring the percentage of [¹⁴C]-AMPA HPLCchromatogram peak area converted to some other [¹⁴C]-compound, such asN-acetyl-[¹⁴C]-AMPA, with the amount of N-acetyl-[¹⁴C]-AMPA producedduring the reaction in which [¹⁴C]-AMPA and 1 mM acetyl-CoA aresubstrates for PhnO being established as the 100% reference. The resultsare shown in Table 12.

TABLE 12 AMPA Transacylase Enzyme Efficiency for Acyl-CoA Acyl DonorSubstrate Acyl-CoA Acyl Donor [¹⁴C]-AMPA % Conversion¹ % ActivityAcetyl-CoA 0.1 mM 79.2 79.2 Acety-CoA 0.5 mM 98.7 98.7 Acety-CoA 1 mM100.00 100.00 Propionyl-CoA 0.1 mM 78.2 78.2 Propionyl-CoA 0.5 mM 97.897.8 Propionyl-CoA 1 mM 100.00 100.00 Glutaryl-CoA 0.1 mM 0.81 0.81Glutaryl-CoA 0.5 mM 0.00 0.00 Glutaryl-CoA 1 mM 0.57 0.57Methylmalonyl-CoA 0.1 mM 1.11 1.11 Methylmalonyl-CoA 0.5 mM 2.08 2.08Methylmalonyl-CoA 1 mM 2.21 2.21 Crotonoyl-CoA 0.1 mM 0.80 0.80Crotonoyl-CoA 0.5 mM 0.00 0.00 Crotonoyl-CoA 1 mM 0.00 0.00Isobutyryl-CoA 0.1 mM 2.10 2.10 Isobutyryl-CoA 0.5 mM 0.20 0.20Isobutyryl-CoA 1 mM 0.00 0.00 Succinyl-CoA 0.1 mM 5.06 5.06 Succinyl-CoA0.5 mM 3.38 3.38 Succinyl-CoA 1 mM 1.56 1.56 Tiglyl-CoA 0.1 mM 0.00 0.00Tiglyl-CoA 0.5 mM 0.00 0.00 Tiglyl-CoA 1 mM 0.99 0.99 Valeryl-CoA 0.1 mM0.24 0.24 Valeryl-CoA 0.5 mM 0.00 0.00 Valeryl-CoA 1 mM 0.33 0.33Desulfo-CoA 0.1 mM 0.95 0.95 Desulfo-CoA 0.5 mM 1.25 1.25 Desulfo-CoA 1mM 0.52 0.52 ¹percentage of [¹⁴C]-AMPA HPLC chromatogram peak areaconverted to some other [¹⁴C]-compound, such as N-acetyl-[¹⁴C]-AMPA,with the amount of N-acetyl-[¹⁴C]-AMPA produced during the reaction inwhich [¹⁴C]-AMPA and 1 mM acetyl-CoA are substrates for PhnO beingestablished as the 100% reference

These results indicate that PhnO enzyme is capable of efficientlyutilizing acyl-CoA associated compounds which have an acyl group with acarbon chain length of not more than three for transacylating AMPA.Other compounds which have a longer carbon chain length than propionyl-and which are not broad or bulky, such as methylmalonly-, isobutyryl-,and succinyl-CoA compounds are also effective acyl-CoA acyl donors, butat a lower enzyme efficiency.

Example 7

This example illustrates the in vitro expression and targeting of anAMPA acyltransferase protein into isolated chloroplasts.

Many chloroplast-localized proteins are expressed from nuclear genes asprecursors and are targeted to the chloroplast by a chloroplast transitpeptide (CTP). The CTP is removed during steps involved in import of thetargeted protein into the chloroplast. Examples of such chloroplastproteins include the small subunit (SSU) of ribulose-1,5-bisphosphatecarboxylase (RUBISCO), 5-enol-pyruvylshikimate-3-phosphate (EPSPS),ferredoxin, ferredoxin oxidoreductase, the light-harvesting-complexprotein I and protein II, and thioredoxin F. It has been demonstrated invivo and in vitro that non-chloroplast proteins may be targeted to thechloroplast by use of fusions with a CTP and that a CTP sequence issufficient to target a protein to the chloroplast (Della-Cioppa et al.,1987). 5-enolpyruvylshikimate-3-phosphate synthetase (EPSPS) enzyme islocated in the chloroplast and is the glyphosate target in plants.Targeting glyphosate oxidoreductase to the chloroplast has been found toprovide tolerance to plants to glyphosate, although GOX localized to thecytoplasm is also able to provide such tolerance. Generally, recombinantGOX enzyme is localized to the chloroplast. GOX mediated glyphosatemetabolism produces AMPA, which has been shown to be phytotoxic. It hasbeen shown herein that PhnO is capable of AMPA N-acylation and thatN-acetyl-AMPA is not phytotoxic. Therefore, it may be necessary toinactivate AMPA in plants. This assumes that AMPA acyltransferase can beexpressed in plants as an active enzyme, and that such acyltransferasesare capable of being imported into the chloroplast and retain enzymaticactivity. In view of the AMPA phytotoxicity as described in example 1,an AMPA acyltransferase gene was introduced into plant expressionvectors to test expression in plants. In addition, import ofacyltransferase into chloroplasts was also tested.

A DNA sequence encoding a chloroplast targeting peptide was linked 5′ toand in frame with a DNA sequence encoding an AMPA acyltransferase. A DNAsequence encoding an arabidopsis ribulose-1-bis-phosphate carboxylasesmall subunit chloroplast transit peptide (CTP, SEQ ID NO:9) was excisedfrom pMON17058 using BglII and NcoI restriction endonucleases, andinserted into complementary restriction sites in pMON15028 to producepMON15029, so that the CTP coding sequence was linked 5′ to and in framewith the phnO coding sequence in pMON15028. The resulting chimeric phnOgene in pMON15029 is capable of producing a chloroplast targeted PhnOprotein. An EcoRI to BglII DNA cassette containing the CTP-PhnO codingsequence, SEQ ID NO:10, from pMON15029 was inserted into EcoRI and BamHIsites in pBlueScript KS(−) to produce pMON15036. The CTP-PhnO codingsequence in pMON15036 can be expressed in an in vitrotranscription/translation system from a phage T3 promoter. A similarplant transient expression plasmid, pMON15035, was constructed, butwithout the chloroplast targeting sequence. An EcoRI to BglII DNAfragment containing only the phnO coding sequence was excised frompMON15028 and inserted into EcoRI and BamHI sites in pBlueScript KS(+)so that PhnO could be produced from a phage T7 promoter in an in vitrotranscription/translation system. An NcoI to EcoRI DNA sequence encodingPhnO was excised from pMON15028 and inserted into pMON17061, producingpMON15032. pMON15032 provides for expression of phnO from an E. colirecA promoter. A BglII to EcoRI DNA fragment encoding PhnO was excisedfrom pMON15028 and inserted into pBlueScript SK(−) to produce pMON15033.pMON15033 provides for expression of phnO from an E. coli lac promoter.A BglII to EcoRI DNA fragment encoding CTP-PhnO was excised frompMON15029 and inserted into compatible sites in pBlueScript SK(−),providing for expression of chloroplast targeted PhnO protein from an E.coli lac promoter from pMON15034.

pMON15032, pMON15033, and pMON15034 were introduced into E. coli JM101.Cultures were grown and induced as described above, except thatexpression from cells containing pMON15032 was induced with addition of50 parts per million nalidixic acid in 0.1 M NaOH. Cleared lysates wereprepared from each culture and subjected to an AMPA acyltransferaseassay as described above in order to determine the presence of AMPAacyltransferase activity. All lysates contained substantial amounts ofacyltransferase activity above control levels. More importantly, theCTP-PhnO peptide (SEQ ID NO:12) expressed from pMON15034 appeared toretain full enzymatic acyltransferase activity.

pMON15035 (PhnO) and pMON15036 (CTP-PhnO) were used in vitro to generate[³⁵S]-methionine labeled PhnO protein for use in a chloroplast importassay. Briefly, the procedure used for in vitro transcription andtranslation was as described in Short Protocols In Molecular Biology,Third Edition, Ed. Ausubel et al., Wiley & Sons Pub., (1995), which isherein incorporated by reference. About 20 micrograms of plasmid DNA wasdigested to completion with HindIII restriction endonuclease in a 100microliter reaction. 20 microliters of the plasmid digest, or about 4micrograms of linearized plasmid DNA, was used in an in vitrotranscription reaction to generate mRNA for producing PhnO or CTP-PhnOprotein product in later translation reactions. Transcription reactionsconsisted of 20 microliters of linearized plasmid DNA, 20 microliters ofa 5× transcription buffer (200 mM TrisHCl pH 8.0, 40 mM MgCl₂, 10 mMspermidine and 250 mM NaCl), 20 microliters of 5× ribonucleosidetriphosphate mix (5 mm each ATP, CTP, UTP, 5 mM diguanosine triphosphate(G-5′ppp5′-G)TP, 5 mM GTP), 10 microliters 0.1 M dithiothreitol (DTT),10 microliters RNasin™ (a pancreatic ribonuclease inhibitor mixture fromPromega), 4 microliters RNA polymerase (T7 or T3, New England Biolabs,Inc.), and distilled, deionized water to 100 microliters. Each reactionwas incubated at 37° C. for one hour. 4.5 microliters of each reactionwas analyzed on a 1.4% agarose formaldehyde gel to ensure that eachreaction produced adequate RNA template for the following translationstep.

20 microliters of the transcription reactions were used for producing[³⁵S]-methionine labeled PhnO proteins for use in a chloroplast importassay. Briefly, RNA was mixed with 6 microliters of an aqueous aminoacid mixture without methionine, 15 microliters of [³⁵S]-methionine(1400 Ci/mmol, Amersham), and 200 microliters of a rabbit reticulocytelysate. These reactions were incubated at 37° C. for two hours andplaced on dry ice for storage. A 10 microliter sample of each reactionwas analyzed on a 15% SDS-PAGE gel. Gels were vacuum dried and placeddirectly onto the emulsion side of KODAK™ X-O-MAT™ film forautoradiography. The results indicated that each plasmid producedrespective peptides of predicted molecular mass for PhnO (pMON15035) andCTP-PhnO (pMON15036) in sufficient quantity to test for uptake intochloroplasts in an import assay.

Intact chloroplasts were isolated from one head of deveined Romainelettuce according to Edelman et al., Methods in Chloroplast MolecularBiology, Elsevier Biomedical Press, Chap. 86, 1982. One liter ofgrinding buffer (GR-buffer) stock was prepared (2 mM NaEDTA, 1 mM MgCl2,1 mM MnCl2, 50 mM Hepes-KOH pH 7.5, and 0.33 mM sorbitol). Immediatelybefore use, 890 mg of ascorbic acid was added to 900 ml of GR-bufferstock solution. One head of torn, deveined Romaine lettuce was mixedwith 900 ml GR-buffer and emacerated by mixing in a Waring blender threetimes for three seconds each time at high speed. The slurry was filteredthrough four layers of Miracloth, and the filtrate was centrifuged at5,000 RPM for 10 minutes at 4° C. in a SORVALL™ GS-3 rotor. Thesupernatant was decanted and the pellet resuspended with a glass rod in4 milliliters of GR-buffer. Chloroplasts were isolated by centrifugationthrough a Percoll gradient. 80% Percoll was prepared by mixing 16 mls ofPBF-Percoll with 4 mls of 5× Buffer (10 mM EDTA, 5 mM MgCl₂, 5 mM MnCl₂,250 mM Hepes-KOH, 30 grams sorbitol, 490 mg NaAscorbate, 85.5 mgglutathione to 100 mls with ddH₂0). A 40% Percoll solution was preparedby combining 8 mls PBF-Percoll with 4 mls 5× Buffer and 8 mls of ddH₂0.A Percoll gradient was prepared in a 30 ml Corex tube by layering 10 mlsof 40% Percoll onto 10 mls of 80% Percoll. Chloroplasts were isolated bylayering the resuspended chloroplasts onto the percoll gradient,spinning at 9,500 RPM for ten minutes in an SS-34 SORVALL™ swingingbucket rotor at 4° C. for ten minutes with the brake on. Brokenchloroplasts remain in the upper layer and were pipetted off. The intactchloroplasts were located at the interface of the 40/80% Percollgradient and were removed to a new 30 ml COREX™ tube. The isolatedchloroplasts were washed two times with GR-buffer and centrifuged forcollection after each wash in a SS-34 rotor at 6,000 RPM for ten minutesat 4° C. with the brake off. Isolated, washed chloroplasts wereresuspended in 1 ml sterile 50 mM Hepes-KOH pH 7.7, 330 mM sorbitol bygently stirring with a glass rod, and the chlorophyll concentration ofthe slurry was determined. 5 mls of an 80% acetone solution was added to20 microliters of the chloroplast slurry and vortexed gently. Theresulting mixture was filtered through a Whatman™ #1 filter paper into aculture tube. The absorbance of the filtrate was determined at 645 nmand 663 nm against an 80% acetone blank. The chlorophyll concentrationin micrograms per ml was determined according to equation #1 as[chlorophyll μg/ml]=[A₆₄₅+[A₆₆₃*(8.02)]. The mass of the chlorophyll inμg is calculated by taking the amount of chlorophyll measured in μg/mland multiplying by the volume into which the chloroplasts wereresuspended (equation #2), which is 5 mls in this example. Thus, theconcentration of chlorophyll in μg/μl in the measured sample isequivalent to the value determined in equation #2 divided by the volumeof the sample measured, which in this example is 20 μl. In this example,A₆₄₅ was determined to be 0.496, and A₆₆₃ was determined to be 1.0814.Thus, the concentration of chlorophyll in the measured sample was 4.67μg/μl. The concentration of chlorophyll in the chloroplast slurry wasadjusted to 4.0 μg/μl with Hepes-KOH pH 7.7, 330 mM sorbitol solutionand the resulting chloroplast suspension was stored on ice in the dark.

A typical 300 microliter uptake experiment contained 5 mM ATP, 8.3 mMunlabeled methionine, 322 mM sorbitol, 58.3 mM Hepes-KOH (pH 8.0), 50microliters reticulocyte lysate translation products, and intactchloroplasts (about 200 microgram chlorophyll). The uptake mixtures weregently rocked at room temperature in 10×75 mm glass tubes, directly infront of a fiber optic illuminator set at maximum light intensity usinga 150 Watt bulb. Two separate 70 microliter samples of each uptake mixwere removed at 0, 5, 10 and 15 minutes. One sample was centrifuged over100 microliter silicone-oil gradients in 150 microliter polyethylenetubes by centrifugation at 11,000×g for 30 seconds, and immediatelyfrozen in dry ice. Under these conditions, the intact chloroplasts forma pellet under the silicone-oil layer and the incubation mediumcontaining the reticulocyte lysate remains floating on the surface ofthe interface. The other sample was treated with protease (one tenthvolume or 7 microliters of 0.25 mg/ml each trypsin and chymotrypsinprotease mixture) for thirty minutes on ice, then subjected tosilicone-oil separation and frozen on dry ice. The chloroplast pelletswere then resuspended in 50-100 microliters of a lysis buffer (10 mMHepes-KOH pH 7.5, 1 mM PMSF, 1 mM benzamidine, 5 mM ε-amino-n-caproicacid, and 30 micrograms per ml aprotinin) and centrifuged at 15,000×gfor 20 minutes to pellet the thylakoid membranes. The clearedsupernatant (stromal proteins) from this spin, and an aliquot of thereticulocyte lysate incubation medium from each uptake experiment, weremixed with an equal volume of 2×SDS-PAGE sample buffer and analyzed on a15% SDS-PAGE gel, dried, and exposed to film as described above.Chloroplasts exposed to [³⁵S]-methionine labeled CTP-PhnO contained[³⁵S]-labeled protein of a size consistent with the predictedCTP-processed form of PhnO, while chloroplasts exposed to methioninelabeled PhnO were devoid of labeled protein. Labeled protein importedinto the chloroplasts was also protease resistant. These resultsindicated that PhnO could be targeted to chloroplasts when fused to aplastid targeting peptide sequence.

Example 8

This example illustrates the identification and characterization ofplants transformed with an AMPA acyltransferase.

A wide variety of plant species have been successfully transformed usingany number of plant transformation methodologies well known in the art.In particular, Agrobacterium tumefaciens mediated plant transformationis the preferred method presently in use, however, ballistic methodswhich increase delivery of naked DNA directly to plant cells throughmicroprojectile bombardment are also very effective in producingrecombinantly transformed plants. In addition, methods which involve theuse of liposomes, electroporation, chemicals that increase free DNAuptake, and transformation using viruses or pollen are alternativeswhich can be used to insert DNA constructs of this invention into plantcells. Plants which can be transformed by the practice of the presentinvention include but are not limited to corn, wheat, cotton, rice,soybean, sugarbeet, canola, flax, barley, oilseed rape, sunflower,potato, tobacco, tomato, alfalfa, lettuce, apple, poplar, pine,eucalyptus, acacia, poplar, sweetgum, radiata pine, loblolly pine,spruce, teak, alfalfa, clovers and other forage crops, turf grasses,oilpalm, sugarcane, banana, coffee, tea, cacao, apples, walnuts,almonds, grapes, peanuts, pulses, petunia, marigolds, vinca, begonias,geraniums, pansy, impatiens, oats, sorghum, and millet. DNA moleculesfor use in the present invention can be native or naturally occurringgenes or chimeric genes constructed from useful polynucleotide sequencesincluding promoters, enhancers, translated or non-translated leaders,sequences encoding signal peptides, sequences encoding transit peptides,structural genes, fusions of structural genes, terminators, introns,inverted repeats or direct repeats, linkers, and polyadenylationsequences. DNA sequences contemplated in this invention include singleand double stranded polynucleotide sequences, linear sequences, andcovalently closed circular polynucleotide sequences, plasmids, bacmids,cosmids, bacterial artificial chromosomes (BAC's), yeast artificialchromosomes (YAC's), and viral DNA and RNA sequences. In considerationof Agrobacterium mediated plant transformation, suitable planttransformation vectors include those derived from a Ti plasmid ofAgrobacterium tumefaciens, as well as those disclosed, for example byHerrera-Estrella (1983), Bevan (1984), Klee (1985) and EPO publication120,516 (Schilperoort et al.). In addition to plant transformationvectors derived from the Ti or root-inducing (Ri) plasmids ofAgrobacterium, alternative methods as described above can be used toinsert the DNA constructs of this invention into plant cells.

Plasmids used for plant transformation generally were constructed fromvectors which have been described elsewhere, particularly in U.S. Pat.No. 5,463,175 (Barry et al., 1995), which is herein incorporated byreference. Plasmids were constructed and maintained in E. coli using Tn7aminoglycoside adenylyltransferase resistance (aad gene, commonlyreferred to as streptomycin/spectinomycin or Spc/Str resistance), whichis also a determinant for selection and maintenance in Agrobacterium.Other plasmid maintenance and selectable markers well known in the artfor use in E. coli were also used, consisting essentially of neomycinphosphotransferase, gentamycin acetyltransferase, and beta lactamasegenes alone or present in combination on a single replicon or vector.Plasmids generally contain oriV, a replication origin derived from thebroad host range plasmid RK2, and ori322 and bom (origin of replicationfor maintenance in E. coli, and basis of mobility for conjugationaltransfer respectively) sequences derived from plasmid pBR322.

A phnO gene encoding an AMPA acyltransferase was inserted intoexpression cassettes in plant transformation vectors. These cassettesgenerally contain the following elements in sequential 5′ to 3′ order: asequence comprising a plant operable promoter, a sequence encoding achloroplast or plastid transit peptide, a cloning site or sitescontained within a polylinker, and a plant functional 3′ nontranslatedregion. Expression cassettes often are constructed to contain uniquerestriction sites flanking the cassette domain so that the entirecassette can be excised from one plasmid and placed into other similarlyconstructed plasmid vectors. Restriction sites comprised of eight basepair recognition sequences are preferred, and most cassettes in thepresent invention are flanked at least on one end by a NotI restrictionendonuclease recognition site. Preferred promoters are the figwortmosaic virus promoter, P-FMV (Gowda et al., 1989), the cauliflowermosaic virus 35S promoter CaMV 35S (Odell et al., 1985), or the enhancedCaMV 35S promoter (U.S. Pat. No. 5,196,525; Kay et al., 1987). A numberof other promoters which are active in plant cells have been describedin the literature. Such promoters may be obtained from plants or plantviruses and include, but are not limited to the nopaline synthase (NOS)and octopine synthase (OCS) promoters which are carried ontumor-inducing plasmids generally found within virulent and non-virulentstrains of Agrobacterium tumefaciens, the cauliflower mosaic virus(CaMV) 19S promoter, the comalina yellow mottle virus promoter, thesugar cane bacilliform DNA virus promoter, the peanut chlorotic streakvirus promoter, the rice actin promoter, and the light-inducibleribulose 1,5-bisphosphate carboxylase small subunit promoter(ssRUBISCO). These promoters can used to create various types of DNAconstructs useful for gene expression in plants (see for example Barryet al. U.S. Pat. No. 5,463,175). Particularly desirable promoters whichare contemplated because of their constitutive nature are theCauliflower Mosaic Virus 35S (CaMV35S) and the Figwort Mosaic Virus 35S(FMV35S) promoters which have previously been shown to produce highlevels of expression in most plant organs. Other promoters which woulddirect tissue specific or targeted expression are also contemplated, forexample in tissue such as leaves, meristem, flower, fruit and organs ofreproductive character. In addition, chimeric promoters are alsoenvisioned. Nopaline synthase gene (NOS 3′) and the pea ribulosebisphosphate carboxylase synthase E9 gene (E9 3′) 3′ nontranslatedtermination and polyadenylation sequences were also used.

Expression cassettes consisting of a AMPA acyltransferase structuralgene inserted downstream of a promoter and between a sequence encoding achloroplast targeting peptide and a 3′ nontranslated sequence weregenerally present on a plant transformation vector. Expression cassetteswere generally flanked on either end of the cassette by a nopaline typeT-DNA right border region on one end and a left border region on theother end, both border regions derived from pTiT37 (Fraley et al.,1985). Some plant transformation vectors only contained the right borderregion, required for initiation of T-DNA transfer from Agrobacterium tothe host cell. Most plant transformation vectors also contained a GOX(glyphosate oxidoreductase) gene, as described above, and in U.S. Pat.No. 5,463,175. GOX enzyme expressed from these vectors was generallytargeted to the chloroplast when inserted into the plant genome.

Plant transformation vectors were mobilized into the ABI Agrobacteriumstrain A208 carrying the disarmed Ti plasmid pTiC58 (pMP90RK) (Koncz andSchell, 1986). The Ti plasmid does not carry the T-DNA phytohormonegenes which induce crown gall formation. Mating of the plant vector intoABI was done by the triparental conjugation system using the helperplasmid pRK2013 (Ditta et al., 1980). Alternatively, the planttransformation plasmid can be introduced into the ABI strain byelectroporation as described by Mattanovich et al. (Efficienttransformation of Agrobacterium spp. by electroporation, Nucleic AcidsRes. (1989), 17(16), 6747), which is herein incorporated by reference.When plant tissue is incubated with the ABI::plant vector conjugate, therecombinant vector is transferred to the plant cells by the virfunctions encoded by the disarmed pTiC58 plasmid. Ideally, therecombinant vector opens at the T-DNA right border region, and the DNAbetween the right and left border sequences is transferred directionallyand inserted into the host plant genome, although the entire recombinantplant transformation vector sequence may be transferred and inserted.The pTiC58 Ti plasmid does not transfer to the plant cells but remainsin the Agrobacterium donor.

Recombinant plants can be regenerated from plant cells or plant tissuewhich has been transformed with a functional AMPA acyltransferasestructural gene. The choice of methodology for the regeneration step isnot critical, with suitable protocols being available for hosts fromLeguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot,celery, parsnip), Cruciferae (cabbage, radish, rapeseed, etc.),Cucurbitaceae (melons and cucumber), Gramineae (wheat, rice, corn,etc.), Solanaceae (potato, tobacco, tomato, peppers), and various floralcrops. See for example, Ammirato, 1984; Shimamoto, 1989; Fromm, 1990;and Vasil, 1990). Recombinant plants which have been transformed with anAMPA acyltransferase can also be selected on medium containing AMPA. Theappropriate inhibitory concentration of AMPA can readily be determinedby one of ordinary skill in the art for any particular host by screeningfor AMPA toxicity as described in example 1. Alternatively, when AMPAacyltransferase is transformed into plants previously transformed withGOX and selected for growth on glyphosate, either AMPA or glyphosate canbe used as the selective ingredient for selecting for transformationevents which express sufficient levels of AMPA acyltransferase enzyme.Glyphosate must be applied at levels which would otherwise be inhibitoryto a recombinant plant expressing GOX and selected for growth onglyphosate, due to the increased level of AMPA which may be produced asa result of GOX mediated glyphosate degradation. In plants which expressrecombinant GOX enzyme, exposure to increasing levels of glyphosate hasbeen shown to induce yellowing or chlorosis of the leaves, stuntedgrowth characteristics, and infertility. AMPA acyltransferase expressedcoordinately or in combination with GOX expression can overcome thesedetrimental effects. It is also possible to use AMPA as a planttransformation selectable marker as an alternative to glyphosateselection.

Tobacco

Tobacco plants were transformed with a phnO gene. A tobacco leaf disctransformation procedure employed healthy tissue from a leaf of aboutone month old. After a 15-20 minute surface sterilization with 10%CLOROX™ plus a surfactant, leaves were rinsed three times in sterilewater. Leaf discs were punched with a sterile paper punch, and placedupside down on MS104 media (4.3 g/l MS salts, 30 g/l sucrose, 2 ml/l500× B5 vitamins, 0.1 mg/l NAA, and 1.0 mg/l BA), and pre-cultured forone day. Discs were then inoculated with an 1:5 diluted overnightculture of disarmed Agrobacterium ABI containing the subject vector(final culture density about 0.6 OD as determined at 550 nm). Theinoculation was done by placing the discs in sterile centrifuge tubesalong with the culture. After thirty to sixty seconds, the liquid wasdrained off and the discs were blotted between sterile filter paper. Thediscs were then placed upside down on a filter disc on MS104 feederplates and incubated for 2-3 days. After this co-culture period, thediscs were transferred, still upside down, to selection platescontaining MS104 media. After 2-3 weeks, callus formed, and individualclumps were separated from the leaf discs. Shoots were cleanly cut fromthe callus when they were large enough to distinguish from stems. Theshoots were placed on hormone-free rooting media (MSO: 4.3 g/l MS salts,30 g/l sucrose, and 2 ml/1500× B5 vitamins) with selection. Roots formedin 1-2 weeks. Any leaf callus assays are preferably done on rootedshoots while still sterile. Rooted shoots were placed in soil and weremaintained in a high humidity environment (ie: plastic containers orbags). The shoots were hardened off by gradually exposing them toambient humidity conditions.

Three tobacco transformation events, designated as lines 33476, 36779,and 37235 were selected for further analysis. pMON17226 (Barry et al.,U.S. Pat. No. 5,463,175, 1995) was used to produce plant line 33476which contains an FMV-CTP-GOX gene construct. Lines 36779 and 37235 wereproduced using pMON17261, which is a plasmid derived from pMON17226which contains NotI cassette containing an FMV-CTP-PhnO gene sequence(SEQ ID NO:11) in addition to FMV-CTP-GOX. The NotI cassette wasconstructed as follows. The sequence encoding CTP, represented by SEQ IDNO:9, was excised from pMON17058 as a BglII to NcoI fragment andinserted into pMON15028, forming a sequence represented by SEQ ID NO:11in which the CTP coding sequence was upstream of and in frame with thePhnO coding sequence represented within SEQ ID NO:7. The resultingconstruct was designated as pMON15029. The CTP-PhnO coding sequence wasexcised from pMON15029 on a BglII to SacI fragment and combined withpMON17063 fragments to produce pMON15038. pMON17063 was disassembledusing restriction digestion to provide parts necessary for pMON15038construction. pMON17063 was digested with SacI and HindIII to produce avector backbone into which a promoter fragment and the CTP-PhnO sequencewere inserted. pMON17063 was also digested in a separate reaction withHindIII and BglII to produce a fragment containing an FMV promotersequence. The promoter fragment and the CTP-PhnO fragment were ligatedtogether in a reaction along with the vector backbone fragment toproduce pMON15038, containing a NotI cassette harboring a sequenceencoding a chloroplast targeted PhnO peptide expressed from an FMVpromoter and flanked downstream by a NOS E9 3′ transcription terminationand polyadenylation sequence. This NotI sequence was excised frompMON15038 and inserted into the unique NotI site in pMON17241 to producepMON17261, containing a chloroplast targeted GOX coding sequenceexpressed from an FMV promoter and flanked downstream by an E9 3′sequence, along with the CTP-PhnO coding sequence and expressioncassette. Transformation events derived from this vector are expectednot only to be resistant to glyphosate, but to provide resistance toAMPA phytotoxicity as well. Lines 36779 and 37235 derived from pMON17261were analyzed for the presence of genes encoding glyphosateoxidoreductase and AMPA acyltransferase by PCR, for the presence of GOXand PhnO enzymes by western blot, and for the presence of metabolitesproduced as a result of GOX mediated [¹⁴C]-glyphosate degradation byHPLC.

Line 33476, obtained as a transformation event derived from pMON17226,was selected as a “GOX only” control. Lines 36779 and 37235 demonstrateddifferent phenotypes upon exposure to glyphosate and were selected asglyphosate resistant events arising after transformation with pMON17261.Line 37235 became bleached or yellowed upon exposure to glyphosate,similar in phenotype to the GOX only line 33476. However, line 36779displayed no such bleaching effect. DNA was extracted from leaf tissuefor each of these events as well as from wt Samsun tobacco leaf, andsubjected to PCR to determine the presence or absence of thetransforming phnO gene.

Genomic DNA isolated from transformed tobacco lines was used as thetemplate DNA in a PCR reaction and reaction products were compared towild type Samsum tobacco. PCR reactions consisted of 50 microliterstotal volume containing 10× amplification buffer, 1.5 mM MgCl₂,deoxynucleotide mix with each at 1 mM, 50-100 ng genomic DNA, primerseach at a final concentration of 16.8 pM, and 1.5 units of AmpliTaq DNApolymerase (Cetus/Perkin Elmer). Primers (synthesized to order byGENOSYS) consisted of the sequences as set forth in SEQ ID NO:21 and SEQID NO:22. SEQ ID NO:21 is a 20 base pair sequence capable of priming thesynthesis of the P2A phnO gene sequence (SEQ ID NO:7) and hybridizes tothe first twenty nucleotides of the coding sequence in that gene. SEQ IDNO:22 is also a 20 base pair sequence, but is capable of primingsynthesis of a phnO gene from the terminal coding sequence into thestructural coding region and hybridizes to the terminal twentynucleotides of the sequence encoding PhnO. Amplification conditionsconsisted of three cycles of 97° C. for one minute, 60° C. for twominutes, and 72° C. for two minutes, followed by 37 cycles of 94° C. forone minute, 60° C. for two minutes, and 72° C. for two minutes, followedgenerally by a 4° C. soak. 10 microliter samples were generally analyzedby 1% TAE agarose gel electrophoresis to resolve the relevant bands fromresidual primers. Upon ethidium bromide staining of the product gels, aphnO gene amplification product about 432 base pairs as judged by themigration position versus HindIII digested lambda molecular weightmarkers appeared only in the line 33779 extracts, indicating thepresence of the phnO gene in that line.

Seed from Ro transformation events were obtained after self crossing ingrowth chamber conditions. Ro seed were cured and planted to generate R1progeny. Source leaves of R1 progeny at the five leaf stage were exposedto [¹⁴C]-glyphosate by spotting a 2 microliter sample onto each vein (50microliters of [¹⁴C]-glyphosate Na+ salt, 517,000 dpm/microgram, 0.42microgram/microliter mixed with 10 microliters of glycerol). Each leafreceived several spots depending on the number of veins on that leaf.Three days later 15 additional 2 microliter spots were applied to eachleaf. Two weeks later, five 2 microliter spots were applied to each oftwo leaves on each plant. These were new leaves and were not the olderleaves to which glyphosate was initially applied. Five days after thislast application, about 300 milligrams of tissue was sampled from twosink leaves on each plant. The samples from each plant were homogenizedin separate 1 ml volumes of deionized water, centrifuged at 9,000 RPM ina microcentrifuge, and the aqueous volumes were collected and stored onice. Extracts were analyzed by HPLC for the presence of [¹⁴C] labeledmetabolites as in Example 2. The extract obtained from line 33476 (GOX)contained only [¹⁴C]-AMPA. The extract obtained from line 37235contained non-metabolized [¹⁴C]-glyphosate as well as a trace butmeasurable amount of [¹⁴C]-AMPA. Only N-acetyl-[¹⁴C]-AMPA was observedin the extract obtained from line 36779. These results are consistentwith the PCR data which indicated that line 36779 contained at least onecopy of the phnO gene. In addition, the lack of a bleaching effect inline 36779 after exposure to glyphosate is consistent with the presenceof functional GOX and PhnO enzymes and the absence of detectable[¹⁴C]-AMPA.

Cotton

A recombinant phnO gene was transformed into Coker 312 variety cotton(Gossypium hirsutum L.). Glyphosate tolerant cotton lines were producedby Agrobacterium mediated plant transformation using double borderbinary plasmid vectors containing either (1) gox, an Achromobacter sp.strain LBAA gene encoding a glyphosate-metabolizing enzyme glyphosateoxidoreductase (GOX), (2) the gox gene and an E. coli phnO gene encodingPhnO, or (3) the gox/phnO double gene construct along with anAgrobacterium strain CP4 gene encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). All vectors arecapable of replication in both Agrobacterium tumefaciens and E. colihosts, and contain an aminoglycoside adenylyltransferase gene (aad)conferring resistance to aminoglycosides such as spectinomycin orstreptomycin and providing a method for plasmid maintenance.

pMON17241 contains a recombinant gene consisting of a 35S FMV promoterlinked 5′ to an Arabidopsis thaliana ribulose-1,5-bisphosphatecarboxylase small subunit (SSU1A) gene sequence encoding a plastid orchloroplast targeting peptide (Timko et al., 1988) which istranslationally fused to a gox gene coding sequence, which is linked 3′to a 3′ untranslated region, designated E9, from a pearibulose-1,5-bisphosphate carboxylase gene.

pMON17213 is a double gene plant transformation vector containingexpression cassettes comprising (1) a 35S FMV promoter linked to asequence encoding an Arabidopsis thaliana EPSPS chloroplast targetingpeptide linked in-frame to a strain CP4 EPSPS coding sequence, which islinked 3′ to an E9 3′ untranslated region; and (2) a 35S FMV promoterlinked to an SSU1A gene sequence encoding a plastid targeting peptidelinked in-frame to a GOX coding sequence, which is linked 3′ to a NOS 3′termination sequence.

pMON17261, described above, is a double gene plant transformation vectorcontaining expression cassettes comprising (1) an FMV 35S promoterlinked to an SSU1A chloroplast targeting peptide coding sequence linkedin-frame to a GOX coding sequence, which is flanked downstream by the E93′ untranslated region; and (2) an FMV 35S promoter linked to an SSU1Achloroplast targeting peptide coding sequence (SEQ ID NO:9) linkedin-frame to a PhnO coding sequence (SEQ ID NO:7), which is linked 3′ toa NOS 3′ sequence.

pMON10151 is a double gene plant transformation vector containingexpression cassettes comprising (1) an FMV 35S promoter linked to anSSU1A chloroplast targeting peptide coding sequence (SEQ ID NO:9) linkedin-frame to a PhnO coding sequence (SEQ ID NO:7), which is flankeddownstream by a NOS 3′ sequence; and (2) an enhanced 35S promoter linkedto an SSU1A chloroplast targeting peptide coding sequence linkedin-frame to a GOX coding sequence which is flanked downstream by a NOS3′ sequence.

pMON10149 is a triple gene plant transformation vector containingexpression cassettes comprising (1) an FMV 35S promoter and a petuniaHSP70 5′ untranslated leader sequence linked to an SSU1A chloroplasttargeting peptide coding sequence linked in-frame to an EPSPS codingsequence, which is flanked downstream by the E9 3′ termination andpolyadenylation sequence; (2) an FMV 35S promoter linked to an SSU1Achloroplast targeting peptide coding sequence (SEQ ID NO:9) linkedin-frame to a PhnO coding sequence (SEQ ID NO:7), which is flankeddownstream by a NOS 3′ sequence; and (3) an enhanced 35S CaMV promoterlinked to an SSU1A chloroplast targeting peptide coding sequence linkedin-frame to a GOX coding sequence, which is flanked downstream by anopaline synthase 3′ polyadenylation sequence (NOS 3′).

Plasmid vectors were assembled in E. coli K12 strains and mated into adisarmed ABI Agrobacterium strain. Aminoglycoside resistantAgrobacterium strains were used to transform Coker 312 derived hypocotylsections with modifications as described by Umbeck et al. (1987) andUmbeck (U.S. Pat. No. 5,159,135 (1992), incorporated herein byreference), except that plants were regenerated with modificationsdescribed by Trolinder and Goodin (1987). Selection for glyphosateresistance produced several lines of cotton callus, which weresubsequently determined by PCR of genomic DNA to contain the respectivegenes encoding EPSPS, GOX or PhnO transferred from Agrobacterium.Additionally, these same callus lines were determined by Western blotanalysis to express the desired genes. After plant regeneration, wholecotton plants which contained the indicated coding sequences wererecovered.

Previously identified plants transformed with a double gene glyphosateresistance cassette comprised of EPSPS and GOX encoding genes weredetermined to be resistant to glyphosate when applied at 48 ounces peracre through the 6-7 leaf stage, however severe bleaching of the leaveswas observed. This phytotoxic effect was presumed to be due to theformation of AMPA as a result of GOX mediated glyphosate degradation. Totest this, AMPA was sprayed at three different rates onto wild typeCoker 312 plants. Leaf chlorosis and stunted growth was observed inplants at four days post-application of glyphosate at 640 ounces peracre and at eight days post-application of 64 ounces per acre. Theseresults suggested that the phytotoxic effect observed in EPSPS/GOXtransformed cotton plant lines was a result of GOX mediated AMPAproduction in plants, and that the phytotoxic effect may be obviated byco-expression of an AMPA acyltransferase along with GOX. To test this,cotton plants expressing GOX or GOX plus EPSPS alone or in combinationwith PhnO expression were treated with [¹⁴C]-glyphosate, and themetabolism of the isotope labeled glyphosate was monitored in leaftissue seven days after application.

Coker 312 glyphosate resistant recombinant cotton line 4416 was selectedas a glyphosate resistant cotton line after transformation withpMON10149, a triple gene Agrobacterium tumefaciens mediated doubleborder plant transformation vector containing chloroplast targetedEPSPS, GOX, and PhnO, each expressed independently from separate 35Spromoters. Several 4416 R3 plants were raised from R2 seed. One leaf ofeach plantlet at the three or four stage was treated with a mixture ofROUNDUP ULTRA™ commercial herbicide mixture (Lot No. GLP-9701-7428-F)which had been fortified with [¹⁴C]-glyphosate (Code No. C-2251). TheROUNDUP ULTRA™ was shown to be 30.25% glyphosate acid by weight and the[¹⁴C]-glyphosate had a radiochemical purity of 97.3% and a specificactivity of 36.36 mCi/mmol. The treatment solution consisted ofapproximately 38 μL containing 1.60×10⁶ dpm with a [¹⁴C]-glyphosatespecific activity of 1.713×10³ dpm/μg glyphosate acid. Three or sevendays after topical application the treated leaves were rinsed withwater, frozen in liquid nitrogen, fractured with a spatula and thenground using a TEKMAR™ tissuemizer in 10 mL of water. The leaf extractswere adjusted to pH 3.5-4.0 with 1N HCl and approximately 4-8000 dpmwere analyzed for the presence of [¹⁴C]-metabolites by HPLC with liquidscintillation vial collection and detection (HPLC/LSC) as described inexample 2. The new growth including the meristem and new leaves thatemerged following topical application were also extracted and analyzedfor [¹⁴C]-metabolites. The results are shown in Table 13.

TABLE 13 [¹⁴C]-Glyphosate Metabolism In Glyphosate Resistant Cotton %[¹⁴C] metabolite in Glyphosate % [¹⁴C] metabolite in Treated LeafExtract . . .* New Growth Extract . . .* Line 4416 N-Acetyl- N-Acetyl-Plant# Glyphosate AMPA AMPA Glyphosate AMPA AMPA MD03 55.2 2.5 37.4 nd**nd 93.4 MD04 94.6 2.1 1.7 97.9 nd nd A01 48.6 2.1 44.7 0.9 0.2 95.8 A0267.3 2.0 29.1 0.7 0.2 96.5 A03 48.8 2.0 43.4 1.2 nd 94.0 A04 19.4 1.673.9 1.5 nd 94.0 A05 59.9 2.2 31.1 2.2 0.2 95.2 A06 38.2 nd 60.9 1.5 0.293.5 A07 64.1 nd 26.8 1.4 0.5 93.9 A08 90.9 2.0 1.9 91.2 2.5 1.9*[¹⁴C]-Glyphosate, [¹⁴C]-AMPA, and N-Acetyl-[¹⁴C]-AMPA as a percentageof total [¹⁴C] isotope observed by HPLC/LSC in each sample. **ndindicates that the metabolite was not detected by HPLC/LSC

Analysis of the water rinsed glyphosate treated leaves indicated thepresence of significant levels of N-acetyl-[¹⁴C]-AMPA in eight of theten plants tested. These levels represented 27-74% of the isotopeextracted from the treated leaves. The remaining activity was almostentirely [¹⁴C]-glyphosate. Very little of the [¹⁴C] isotope was presentas [¹⁴C]-AMPA. The remaining two plants had very limited ability tometabolize glyphosate as indicated by the high levels of[¹⁴C]-glyphosate remaining on or in the leaves. One of these plants alsoshowed signs of stunting seven days after treatment, indicatingglyphosate phytotoxicity.

Analysis of new growth in the ten plants tested showed that thepredominant form of [¹⁴C] labeled metabolite present wasN-acetyl-[¹⁴C]-AMPA at greater than 90% of the total radioisotope in thesamples. In contrast, more than 90% of the isotope in the remaining twoplants was in the form of [¹⁴C]-glyphosate, consistent with the analysisof the extract from the treatment leaf for these two plants.

The metabolism of [¹⁴C]-glyphosate in recombinant cotton lines 4268(GOX/PhnO) and 3753 (EPSPS/GOX) was also studied. Plants in this studywere treated as indicated above for cotton line 4416, by applyingdroplets of ROUNDUP ULTRA fortified with [¹⁴C]-glyphosate to a singleleaf on each plant at the three to four leaf stage. Treated leaves wereharvested and rinsed with water, then ground and extracted, and extractswere analyzed by HPLC as described above for the presence of[¹⁴C]-glyphosate, [¹⁴C]-AMPA, and N-acetyl-[¹⁴C]-AMPA. New growth,including the meristem and new leaves that emerged following applicationwere also extracted and analyzed. The results are shown in Table 14.

TABLE 14 [¹⁴C]-Glyphosate Metabolism In Glyphosate Resistant Cotton *%[¹⁴C] metabolite in Glyphosate *% [¹⁴C] metabolite in Treated LeafExtract . . . New Growth Extract . . . Plant Glyphosate AMPAN-Acetyl-AMPA Glyphosate AMPA N-Acetyl-AMPA GOX/PhnO Plants B01 76.7 3.014.0 3.4 1.0 89.9 B02 63.9 4.8 25.0 1.1 1.5 91.5 B03 54.4 3.2 36.4 0.8nd 94.7 B04 58.3 5.7 28.9 1.1 1.2 91.0 EPSPS/GOX Plants C01 59.8 26.6 nd3.72 85.7 nd C02 92.7 2.1  0.8 92.8 0.8 nd C03 81.2 10.7 nd 13.5 72.0 1.9 C04 86.2 6.4  1.0 13.9 76.2 nd *[¹⁴C]-Glyphosate, [¹⁴C]-AMPA, andN-Acetyl-[¹⁴C]-AMPA as a percentage of total [¹⁴C] isotope labeledmetabolites observed after HPLC/LSC analysis in each sample. **ndindicates that the metabolite was not detected by HPLC/LSC.

Significant levels of N-acetyl-[¹⁴C]-AMPA were present in the treatedleaves of all four line 4268 plants (GOX/PhnO; B01-B04). In contrast,N-acetyl-[¹⁴C]-AMPA was not detectable in extracts obtained from line3753 plants (EPSPS/GOX; C01-C04). Three of these plants containedsignificant levels of [¹⁴C]-AMPA in treated leaf extracts, ranging from6-27%. One line 3753 plant was deficient in the conversion of[¹⁴C]-glyphosate to N-acetyl-[¹⁴C]-AMPA, and this plant also appeared tobe stunted.

90-95% of the [¹⁴C] isotope in extracts of new growth from line 4268plants was determined to be in the form of N-acetyl-[¹⁴C]-AMPA. However,72-86% of the [¹⁴C] isotope in extracts of new growth from three of theline 3753 plants was determined to be [¹⁴C]-AMPA, with [¹⁴C]-glyphosateaccounting for the remainder of the isotope in these tissues. 93% of theisotope obtained from line 3753 plant number C02 was determined to be[¹⁴C]-glyphosate, consistent with the lack of glyphosate metabolism inthe application leaf as well as the observed stunting. In addition,growth regions of all line 3753 plants were discolored and yellowfollowing treatment, but improved with time. By harvest, new growthleaves became mottled.

These results are consistent with the presence of active gox and phnOgene products in the indicated plants. The GOX and PhnO proteins aremetabolizing glyphosate to AMPA and N-acetyl-AMPA in the predictedmanner, and line 4268 plant extracts provide a similar metabolic patternto that observed with line 4416 plant extracts as judged by HPLC and byphenotypic observation. In both lines, the predominant [¹⁴C] product innew growth tissue extracts after [¹⁴C]-glyphosate application isN-acetyl-[¹⁴C]-AMPA. The phytotoxicity as observed by discoloration ofplant leaves in line 3753 after glyphosate application is associatedwith the lack of an AMPA N-acyltransferase activity. In contrast, thepresence of an AMPA N-acyltransferase activity in both the 4416 and the4268 plant lines resulted in a lack of phytotoxic effects observed inline 3753 plants.

Canola

Canola plants were transformed with the vectors pMON17138 and pMON17261and a number of plant lines of the transformed canola were obtainedwhich exhibited glyphosate tolerance. Plants were transformed accordingto the method described in Barry et al. (U.S. Pat. No. 5,633,435).Briefly, Brassica napus cv Westar plants were grown in controlled growthchamber conditions as described. Four terminal internodes from plantsjust prior to bolting or plants in the process of bolting but beforeflowering were removed and surface sterilized in 70% v/v ethanol for oneminute, then in 2% w/v sodium hypochlorite for twenty minutes, thenrinsed three times with sterile distilled deionized water. Stems withleaves attached could be refrigerated in moist plastic bags for up tothree days prior to sterilization. Six to seven stem segments were cutinto 5 mm discs with a Redco Vegetable Slicer 200 maintainingorientation of basal end. Stem discs (explants) were inoculated with 1milliliter of ABI Agrobacterium tumefaciens strain A208 containing arecombinant plant transformation plasmid prepared as described above.Explants were placed basal side down in petri plates containing 0.1×standard MS salts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.7, 1 mg/l BA(6-benzyladenine). The plates were layered with 1.5 ml of mediacontaining MS salts, B5 vitamins, 3% sucrose, pH 5.7, 4 mg/lp-chlorophenoxyacetic acid, 0.005 mg/l kinetin and covered with sterilefilter paper.

Following a 2.3 day co-culture, explants were transferred to deep dishpetri plates (seven explants per plate) containing MS salts, B5vitamins, 3% sucrose, 0.8% agar, pH 5.7, 1 mg/l BA, 500 mg/lcarbenicillin, 50 mg/l cefotaxime, 200 mg/l kanamycin or 175 mg/lgentamicin for selection, and transferred after three weeks to freshmedia, five explants per plate. Explants were cultured in a growth roomat 25° C. with continuous light (Cool White). After an additional threeweeks, shoots were excised from the explants, and leaf recallusingassays were initiated to confirm modification of R₀ shoots. Three tinypieces of leaf tissue were placed on recallusing media containing MSsalts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.7, 5 mg/l BA, 0.5 mg/lnaphthalene acetic acid (NAA), 500 mg/l carbenicillin, 50 mg/lcefotaxime, 200 mg/l kanamycin or gentamicin or 0.5 mM glyphosate. Theleaf assays were incubated in a growth room under the same conditions asexplant culture. After an additional three weeks, the leaf recallusingassays were scored for herbicide tolerance (callus or green leaf tissue)or sensitivity (bleaching).

Each shoot stem was dipped in ROOTONE at the time of excision, placed ina two inch pot containing Metro-MIX 350, and maintained in a closedhumid environment in a growth chamber at 24° C., 16/8 hour photoperiod,400 uE per square meter per second (HID lamps) for a hardening-offperiod of approximately three weeks.

Plasmid pMON17138 is an Agrobacterium mediated single border planttransformation vector maintained in the bacterium by selection onstreptomycin or spectinomycin. pMON17138 contains a single right Tiborder flanking the 3′ end of the genetic elements desired to betransferred into the plant genome. This vector contains two plantoperable expression cassettes. One cassette is comprised of acaulimovirus 35S promoter driving expression of a neomycinphosphotransferase gene (nptII), flanked downstream by a nopalinesynthase 3′ transcription termination and polyadenylation sequence (NOS3′). The other cassette is comprised of a figwort mosaic virus promoter(described in Rogers, U.S. Pat. No. 5,678,319) upstream of a pearibulose bisphosphate carboxylase small subunit transcriptiontermination and polyadenylation sequence. A chloroplast targetedglyphosate oxidoreductase (GOX) coding sequence is inserted between thepromoter and pea 3′ sequence.

Plasmid pMON17261 is an Agrobacterium mediated double border planttransformation vector similar to pMON17138. A chloroplast targeted GOXencoding cassette identical to that in pMON17138 is present downstreamfrom a Ti right border, and upstream of an additional plant operableexpression cassette comprised of a figwort mosaic virus promoter (P-FMV)linked to a NOS 3′ sequence. A chloroplast targeted PhnO coding sequenceis inserted between the second P-FMV and NOS3′ sequences.

R₁ plants derived from transformation events using pMON17261 andpMON17138 were evaluated using a glyphosate spray test described inBarry et al. (U.S. Pat. No. 5,633,435).

Corn

An AMPA acyltransferase gene has also been introduced into Black MexicanSweet corn cells with expression of the gene and glyphosate resistancedetected in callus. Callus tissue was transformed according to themethod described in Barry et al. (U.S. Pat. No. 5,463,175). Variousplasmids were used to introduce glyphosate resistance genes encoding GOXand EPSPS in combination with an AMPA acyltransferase gene into corncells. These plasmids differed from each other with respect to promotersused, chloroplast or plastid targeting peptide sequences used,untranslated leader sequences used, presence or absence of an intron,and type of 3′ terminator used, however all plasmids contained asynthetically derived AMPA acyltransferase gene encoding PhnO containingthe P2A mutation. The synthetic gene was constructed from three smallerpolynucleotide sequences synthesized for Monsanto and characterized forthe presence of the desired DNA coding sequence and amino acid sequencetranslation by Stratagene, Inc., La Jolla, Calif. The non-naturallyoccurring gene was assembled from three smaller sequences comprised ofSEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, wherein the fullyassembled gene is represented by SEQ ID NO:19, and is present in each ofthe plasmids used for the corn callus transformation. The non-naturallyoccurring gene coding sequence was established based on the methoddescribed in Fishhoff et al. in U.S. Pat. No. 5,500,365 in which monocotpreferred codons were used in place of those preferred by E. coli. Thefully assembled gene encodes a full length PhnO protein identical to thenative protein sequence with the exception of the P2A mutationintroduced by PCR using SEQ ID NO:5 and SEQ ID NO:6 to engineerappropriate restriction endonuclease recognition sites into the flankingends of the coding sequence. Plasmids which were used in generating thecorn callus data are shown in Table 15 along with differences withrespect to genetic elements flanking the AMPA acyltransferase encodingsequence.

TABLE 15 Corn Callus Transformation Plasmids and Relevant GeneticElements Plasmid Relevant Genetic Elements* pMON32926[Pe35S/I-Zm•Hsp70/CTP/phnO/T-At•Nos]→ GOX → EPSPS pMON32931[Pe35S/I-Zm•Hsp70/phnO/T-At•Nos] → GOX → EPSPS pMON32932[Pe35S/I-Zm•Hsp70/CTP/phnO/T-At•Nos] → GOX → EPSPS pMON32936[P-Os•Act1/I-Os•Act1/CTP/phnO/T-At•Nos]→ GOX → EPSPS pMON32938[P-Os•Act1/I-Os•Act1/CTP/phnO/T-At•Nos] → GOX → EPSPS pMON32946[Pe35S/L-Ta•Cab/CTP/phnO/T-Ta•Hsp70] ]→ GOX → EPSPS pMON32947[Pe35S/L-Ta•Hsp70/CTP/phnO/T-Ta•Hsp70] → GOX → EPSPS pMON32948 EPSPS→[Pe35S/I-Zm•Hsp70/CTP/phnO/T-At•Nos] → GOX pMON32950EPSPS→[Pe35S/I-Zm•Hsp70/CTP/phnO/T-At•Nos]→ GOX pMON32570 EPSPS→[Pe35S/L-Ta•Cab/I-Os•Act1/CTP/phnO/T-Ta•Hsp70]→GOX pMON32571 EPSPS→[Pe35S/L-Ta•Cab/I-Os•Act1/CTP/phnO/T-Ta•Hsp70] →GOX pMON32572 EPSPS→[Pe35S/L-Zm•Hsp70/I-Os•Act1/CTP/phnO/T-Ta•Hsp70]→GOX pMON32573 EPSPS→[Pe35S/L-Ta•Cab/I-Os•Act1/CTP/phnO/T-Ta•Hsp70]→GOX *Genetic elementscontained within PhnO expression cassettes as indicated in each plasmid.Elements are shown in the order in which they appear in the plasmid,along with the presence of other genes encoding herbicide resistance, ifpresent, flanking the PhnO expression cassette. → indicates thedirection of transcription of each gene or genes flanking the PhnOexpression cassette. Individual elements are described in the text.Promoters which were used included the CaMV e35 S promoter and the riceactin promoter (P-Os.Act1). Introns which were used included thoseobtained from plant genes such as corn Hsp70 (I-Zm.Hsp70) and rice actin(I-Os.Act1). Non-translated leader sequences which were used includedwheat chlorophyll a/b binding protein (L-Ta.Cab) and corn Hsp70(L-Zm.Hsp70). Termination and polyadenylation sequences which were usedincluded Agrobacterium tumefaciens NOS 3′ (T-At.Nos) and wheat Hsp70(T-Ta.Hsp70). The same chloroplast targeting sequence was used in allPhnO expression cassettes, represented by SEQ ID NO: 9.

A [¹⁴C]-glyphosate metabolism assay was used for determining whethertransformed corn callus tissues contain functioning forms of theseenzymes. The assay was developed to screen large numbers of corn callussamples. Callus was obtained from Monsanto Company and Dekalb SeedCompany corn transformation groups. The Monsanto callus samples,individually designated as callus lines “19nn-nn-nn” in Table 16, wereproduced from HI II X B73 corn embryos. Callus samples were bombardedwith complete covalently closed circular recombinant planttransformation vector plasmid DNA or with linear DNA fragments isolatedfrom such plasmids 25-50 days after embryo isolation. Transformed lineswere identified 8-14 weeks after bombardment. These lines weresub-cultured on fresh media every 2 weeks and were 5-7 months old whenused in the metabolism assay. The Dekalb callus lines OO, OR, OW, OX,and OY were obtained from HI II x AW embryos. All line designationscorrespond to the recombinant plasmid or linear fragment used forballistic transformation of callus tissue as noted in the legend toTable 16.

4.5 mCi of N-phosphono-[¹⁴C]-methylglycine ([¹⁴C]-glyphosate) wasobtained from the Monsanto Radiosynthesis group in a 1.5 mM aqueoussolution, having a specific radioactivity of 39.4 mCi/mM (5.2×10⁵dpm/microgram). The sample was identified with code number C-2182.2. Astock solution sterilized by filtration through a 0.2-micron Acrodisk(Gelman no. 4192) was prepared by combining 2.5 mL [¹⁴C]-glyphosate(3.3×10⁸ dpm) with 2.5 mL of corn callus growth medium (N6 medium) and5.0 mg of Mon 0818 surfactant. [¹⁴C]-glyphosate in the resulting dosesolution was 0.75 mM. The N6 medium was described by Chu et al. (1975)and was prepared using salts and vitamins obtained from Sigma ChemicalCompany, St. Louis, Mo. Mon 0818 surfactant is ethoxylated tallowamine,the surfactant used in Roundup herbicide. The dose solution wassubjected to HPLC analysis as described in Example 2. The results areshown in a chromatogram illustrated in FIG. 1. Three radioactive peakswere resolved, the largest of which corresponded to glyphosate (11.3min, 98.8%). Impurity peaks corresponding to [¹⁴C]-AMPA (5.8 min, 0.16%)and an unidentified material (10.2 min, 1.0%) were also present in thedose solution. No peaks corresponding to N-acetyl-[¹⁴C]-AMPA werepresent in the dose solution. Two additional dose solutions wereprepared using these reagents, each of which were scaled three fold to15 ml volumes based on the preparation method described above.

N-acetyl-[¹⁴C]-AMPA was synthesized for use as a retention time HPLCstandard. 1 mL of pyridine and 2 mL of acetic anhydride was added to a20-mL screw cap culture tube and chilled on ice. 0.1 mL of an aqueoussolution of [¹⁴C]-AMPA (6.2×10⁶ dpm, code C-2127.2) was added to thechilled solution. The tube was then removed from the ice bath and warmedto about 50-60° C. A 10-μL sample was removed after about 30 minutes andcombined with 0.5 mL of water and analyzed according to the HPLC methodset forth above. [¹⁴C]-AMPA was not detected, however two newradioactive peaks were identified; one peak at 13.9 minutes (68%) andthe other at 15.4 minutes (32%). A sample of the material eluting at13.9 minutes was isolated and analyzed by negative ion electrospray massspectrometry. The result showed strong ions at m/e 152 and 154, asexpected for this compound, which has a molecular weight of 153 Daltons;the m/z 154 ion was due to the isotopic [¹⁴C]atom. The radioactive peakeluting at 15.4 minutes was not isolated. However, in a separate HPLCexperiment, it was shown to co-elute with syntheticN-acetyl-N-methyl-AMPA. N-methyl-[¹⁴C]-AMPA has previously been shown tobe an impurity in the initial [¹⁴C]-AMPA material.

Under aseptic conditions, corn callus samples were transferred toindividual wells of sterile 48-well COSTAR cell culture clusters (cat.No. 3548). The individual callus samples were not weighed. However, inseveral cases the total weight of the callus samples in a 48-well platewas determined. Typically, the average weight of individual callussamples was approximately 200-250 mg. In each assay, a nontransformedcallus sample, HI II X B73, was included as a control. 50 μL of dosesolution containing 3.3×10⁶ dpm of [¹⁴C]-glyphosate was added to eachcallus sample. 48-well plates were sealed with parafilm and placed in aplastic bag containing a wet paper towel to provide a moist atmosphere.Bags were closed and placed in a dark drawer at 25° C. for 10 days. Eachcallus sample was subsequently transferred to a labeled microcentrifugetube (VWR, 1.7-mL, cat. No. 20170-620). 1.0 mL of de-ionized water wasadded to each tube, and the tubes were closed and placed in round20-tube floating microcentrifuge racks (Nalge cat. no. 5974-1015). Thesemicrofuge tubes were floated in boiling water for 30 minutes, shakenusing a vortex mixer, and centrifuged for 5 minutes using a Fisher brandmicrocentrifuge. 120-μL supernatant samples were removed for analysis byHPLC as described below. The samples were injected using a Waters WISPautoinjector. Chromatographic profiles were obtained for each sampleanalyzed, and quantitative information was obtained by extrapolating thearea under the radioactive elution peaks to total [¹⁴C] in each sample.FIG. 2 shows an HPLC profile of a mixture of standards of the observedradioactive metabolites [¹⁴C] AMPA, [¹⁴C] glyphosate, andN-acetyl-[¹⁴C]-AMPA and the impurity identified asN-acetyl-N-methyl-[¹⁴C]-AMPA.

HPLC analysis was typically completed using a SPHERISORB™ S5 SAX 250mm×10 mm column for most analyses. Some samples were analyzed on anALLTECH™ 5-micron, 250×10 mm SAX column, which provided similarperformance. Two solvents were prepared. Solvent A consisted of 0.005 MKH₂PO₄, adjusted to pH 2.0 with H₃PO₄ and contained 4% methanol. SolventB consisted of 0.10 M KH₂PO₄, adjusted to pH 2.0 with H₃PO₄ and alsocontained 4% methanol. The eluent flow rate was set at 3 mL/min, and thescintillation fluid flow rate was set at 9 mL/min using ATOMFLOW™scintillation fluid (No. NEN-995, from Packard Instruments). All columnsolvent steps were linear, with the injection and column solvent flowrates as indicated in example 2. The column is prepared for anadditional injection at 20 minutes.

Callus samples from 359 transformed corn lines were combined with 50-μLaliquots of [¹⁴C]-glyphosate dose solution and incubated for 10 days inthe dark. Each post-incubation callus sample, together with its clingingdose material, was transferred to a 1.7-mL microcentrifuge tube alongwith 1 mL of water, and each tube was placed in boiling water. This stepcauses cell lysis, releasing soluble intracellular compounds includingany isotope labeled compounds such as glyphosate, AMPA, andN-acetyl-AMPA. It was determined during method development that if thepost-incubation calli were rinsed thoroughly with water, 85-95% of theradioactivity was rinsed off, and HPLC analysis showed that virtuallyall of the radioactivity in the rinses was due to [¹⁴C]-glyphosate andnone was attributable to [¹⁴C]-metabolites. In these experiments, therinsed calli gave extracts containing [¹⁴C]-metabolites in addition to[¹⁴C]-glyphosate. This indicated that the radioactivity in the rinseswas due mainly, if not exclusively, to unabsorbed surface[¹⁴C]-glyphosate. It is important to take this into account whenconsidering the rather low percentages of the dose converted tometabolites, because the percentage calculation includes large amountsof unabsorbed surface radioactivity. The method development work alsoshowed that simply boiling the incubated calli in water released as muchradioactivity as could be released by a conventional grinding/extractingprocedure. Experiments were conducted to show that oiling did not alterthe metabolite profiles. The streamlined procedures made it possible toanalyze large numbers of samples (e.g., 96) at one time. Table 16 showsrepresentative data of the callus samples producing the highest levelsof N-acetyl-[¹⁴C]-AMPA or [¹⁴C]-AMPA obtained after HPLC analysis. Arepresentative chromatogram of a GOX plus AMPA acyltransferasetransformed, glyphosate treated, callus extract sample is shown in FIG.3.

TABLE 16 Transformed Corn Callus Lines Producing Amounts of AMPA orN-Acetyl-AMPA Callus Producing N-Acetyl-[¹⁴C]-AMPA Callus Producing[¹⁴C]-AMPA Transformed Percent** Transformed Percent** Callus* with . .. N-Acetyl-[¹⁴C]-AMPA Callus* with . . . [¹⁴C]-AMPA 1978-05-02 pMON325700.27 1980-28-03 pMON32571 2.89 1978-08-01 pMON32570 0.94 OR523 pMON329262.00 1978-20-02 pMON32570 0.57 OR534 pMON32926 5.00 1978-21-02 pMON325700.23 OR537 pMON32926 2.00 1978-22-01 pMON32570 0.90 OR539 pMON32926 5.081978-24-02 pMON32570 1.80 1971-08-01 pMON32932 2.64 1978-35-01 pMON325700.22 1971-27-03 pMON32932 3.63 1980-01-01 pMON32570 0.27 OO505 pMON329322.73 1980-03-01 pMON32571 0.22 OO509 pMON32932 2.86 1981-28-01 pMON325710.25 OO510 pMON32932 2.34 1981-02-01 pMON32572 0.65 OO512 pMON32932 2.311981-03-01 pMON32572 0.74 OO514 pMON32932 1.98 1981-18-01 pMON32572 0.22OO535 pMON32932 2.88 1981-23-01 pMON32572 0.48 OO538 pMON32932 2.701981-24-02 pMON32572 0.29 OO539 pMON32932 1.97 1981-32-02 pMON32572 1.08OO553 pMON32932 3.56 1977-05-03 pMON32573 0.39 OO576 pMON32932 3.49OR516 pMON32926 1.91 OO579 pMON32932 2.85 1972-14-01 pMON32931 0.401986-17-01 pMON32936 2.29 1972-32-01 pMON32931 0.75 1986-18-03 pMON329363.05 1972-33-01 pMON32931 0.55 1986-18-04 pMON32936 2.15 OO544 pMON329320.28 1986-28-02 pMON32936 2.06 1986-06-01 pMON32936 0.30 1983-12-02pMON32938 2.41 1986-08-01 pMON32936 1.13 1983-31-01 pMON32938 2.901986-08-03 pMON32936 0.70 1985-03-02 pMON32946 2.51 1986-12-01 pMON329360.33 1985-38-01 pMON32947 1.99 1986-18-02 pMON32936 0.40 OX512 pMON329482.43 1986-18-03 pMON32936 0.51 OX533 pMON32948 3.91 1986-18-04 pMON329361.09 OX556 pMON32948 12.11 1986-22-04 pMON32936 0.64 OY504 pMON329502.25 1983-11-01 pMON32938 0.21 OY511 pMON32950 2.53 OW534 pMON32946 0.77OY528 pMON32950 2.58 OW542 pMON32946 0.85 OY532 pMON32950 2.241985-26-01 pMON32947 0.60 OY534 pMON32950 4.02 1985-26-03 pMON32947 0.71OY535 pMON32950 2.34 1985-11-04 pMON32952 0.37 OY540 pMON32950 5.57 *Alllines were transformed using ballistic methods. Lines designated by19xx-yy-zz were transformed with isolated linear fragments of plasmids.Linear fragments were isolated so as to be separate from plasmidbackbone structure. **percent radioactivity detected forN-Acetyl-[¹⁴C]-AMPA or [¹⁴C]-AMPA peaks determined as a fraction of thetotal amount of radioactivity in the sample, including residual[¹⁴C]-glyphosate as described in the text.19 of the 359 callus samples tested produced extracts containingN-acetyl-[¹⁴C]-AMPA at a level distinctly higher than the other callussamples. Callus OR516 was the strongest in this respect and was analyzedfive times during a period of two months, providing values ranging from0.50-4.54% (average 1.91%). The basis for the relatively large spread inthe percentage of N-acetyl-[¹⁴C]-AMPA formed at various times isunknown. In four of the five analyses of OR516, the percentage ofN-acetyl-[¹⁴C]-AMPA present was higher than that of [¹⁴C]-AMPA,indicating an efficient conversion of [¹⁴C]-AMPA to N-acetyl-[¹⁴C]-AMPA.The callus next most efficient in producing N-acetyl-[¹⁴C]-AMPA was1978-24-02, which was the only other callus besides OR516 that containedmore N-acetyl-[¹⁴C]-AMPA than [¹⁴C]-AMPA in its extract. One hundred ofthe 359 callus samples tested produced extracts containing [¹⁴C]-AMPA ata level distinctly higher than other callus samples. OX556 was asuperlative producer of [¹⁴C]-AMPA, yielding more than twice as much ofthe metabolite as any other callus in the study. The control callus, HIII X B73, which contained no inserted genes, produced no detectablelevels of N-acetyl-[¹⁴C]-AMPA and only background levels of [¹⁴C]-AMPA.This result indicates that expression of an AMPA acyltransferase in cornis effective in conversion of AMPA produced as a result of GOX mediatedglyphosate degradation to N-acetyl-AMPA.

Wheat

GOX mediated glyphosate degradation has been shown to produce AMPA, andAMPA has previously been shown to be the source of phytotoxic effects.Therefor, effects of wheat plant exposure to the compounds AMPA orN-acetyl-AMPA was determined as in example 2 in order to observe anywheat sensitivity or insensitivity to either of these compounds. Theobservation of any phytotoxic effects would indicate that GOX mediatedglyphosate metabolism would be detrimental to Triticum species.

Wheat immature embryos were exposed to different concentrations of AMPAand N-acetyl-AMPA in a wheat embryo germination assay. MMSO base mediawas prepared containing 40 grams per liter maltose, 2 grams per literGELRITE™, MS salts, and vitamins. Salts, vitamins, and maltose weredissolved in 3500 ml water and the pH was adjusted to 5.8. 500 ml wasdispensed into a separate bottle along with 1 gram of GELRITE™ andautoclaved for 17 minutes. After the medium had cooled to about 45° C.,AMPA or N-acetyl-AMPA was added to a defined concentration. The mixturewas poured into six square Sundae cups under sterile conditions andallowed to solidify.

Immature wheat embryos were isolated from twenty day old seedlings(after anther formation) and inoculated into each MMSO media. EachSundae cup contained nine immature embryo's. Three separate plates wereused for each concentration of AMPA (0, 0.1, 0.15, 0.2, 0.25, 0.3, and1.0 mM) or N-acetyl-AMPA (0, 0.1, 0.3, 1.0, and 3.0 mM). Sundae cupswere incubated for ten days and the length of roots and shoots weredetermined and compared. The results are shown in Table 17.

TABLE 17 Comparison of AMPA and N-acetyl AMPA on Germinating Shoot andRoot Length Phosphonate Compound Shoot (cm) Root (cm) AMPA (mM) 0.0012.6 ± 2.6 7.0 ± 1.9 0.10 11.7 ± 2.5 8.0 ± 2.0 0.15 11.3 ± 2.1 6.3 ± 1.70.20  9.2 ± 1.8 4.6 ± 2.1 0.25  8.5 ± 1.8 3.1 ± 1.6 0.30  6.6 ± 1.8 2.6± 1.6 1.00  0.9 ± 0.1 0.4 ± 0.1 N-Acetyl-AMPA 0.00 12.6 ± 2.6 7.0 ± 1.90.10 12.0 ± 2.4 5.9 ± 1.4 0.30 11.7 ± 3.5 5.2 ± 1.2 1.00 12.2 ± 3.2 5.4± 1.5 3.00 11.2 ± 2.6 5.9 ± 1.6

AMPA was not substantially inhibitory to growth and elongation ofimmature embryo's at concentrations under 0.2 mM. However,concentrations above 0.2 mM were severely inhibitory to both shoot androot elongation, indicating that AMPA may also be phytotoxic to wheatand, considering the nature of the monocot crop species as a whole,phytotoxic to other monocotyledonous crops as well as turf grasses.Germination of immature embryo's was significantly affected when theAMPA level was higher than 0.20 mM. 1.00 mM AMPA eliminated thegermination of immature embryo's in wheat. In contrast, N-acetyl-AMPAwas not inhibitory to shoots and only mildly inhibitory to rootelongation at any concentrations tested in this experiment. The highestN-acetyl-AMPA concentration tested was greater than ten times theminimal non-inhibitory concentration determined for AMPA. There are nosignificant effects to immature embryo germination when theN-acetyl-AMPA concentration is less than 3.0 mM. This result indicatesthat N-acetylation of AMPA in wheat would prevent AMPA phytotoxicityarising as a result of GOX mediated glyphosate herbicide metabolism.

Recombinant glyphosate tolerant wheat plants were generated according tothe method of Zhou et al. (Plant Cell Reports 15:159-163, 1995).Briefly, spring wheat, Triticum aestivum cv Bobwhite, was used as thetarget transformation line. Stock plants were grown in anenvironmentally controlled growth chamber with a 16 hour photoperiod at800 microJoule per square meter per second provided by high-intensitydischarge lights (Sylvania, GTE Products Corp., Manchester, N.H.). Theday/night temperatures were 18/16° C. Immature caryopses were collectedfrom the plants 14 days after anthesis. Immature embryos were dissectedaseptically and cultured on MMS2 medium, a Murashige and Skoog (Physiol.Plant 15:473-497, 1962) basal medium supplemented with 40 grams perliter maltose and 2 milligrams per liter 2,4-D. In some experiments, CM4medium was used. CM4 medium contains is MMS2 medium, but contains only0.5 milligrams per liter 2,4-D and includes 2.2 milligrams per literpicloram. The immature embryos were cultured at 26° C. in the dark.

Immature embryos were transferred five days after culture initiation toan osmotic treatment CM4 medium containing 0.35 M mannitol four hoursprior to bombardment according to the method of Russell et al. (In VitroCell Devel. Biol., 28P:97-105, 1992). Thirty to forty embryos wereplaced in the center of each plate and bombarded in a DuPont PDS1000apparatus. Plasmid DNA was adsorbed onto 1 μm tungsten particlesaccording to the method of Sanford et al. (Particle Sci. Technol.,5:27-37, 1987). Embryos were bombarded twice at a distance of 13 mm fromthe stopping plate. A 100 μm stainless steel screen was placedimmediately below the stopping plate.

After a 16 hour post bombardment treatment on the osmotic medium, theembryos were transferred to MMS2 or CM4 medium. Following a one weekdelay, the embryos were transferred to the MMS2 or CM4 medium containing2 mM glyphosate. After 9-12 weeks of callus proliferation on theselection medium, calli were transferred to a MMS0.2 regeneration mediumcontaining 0.2 mg/l 2,4-D and 0.1 mM glyphosate. Shoots obtained fromthe regeneration medium were transferred to MMS0 without 2,4-D butcontaining 0.02 mM glyphosate.

Glyphosate tolerant R₀ plants as well as R1 progeny were transferred to15 centimeter diameter pots and grown in an environmentally controlledchamber as described above. Two weeks later, the plants were sprayedwith 3 ml/liter ROUNDUP (41% active ingredient, Monsanto Company) in aspray chamber, which was designed to mimic a field dose application of0.6 kilograms glyphosate per hectare. Damage symptoms were observed andrecorded at different stages following the spraying.

Genomic DNA was isolated from leaf tissue of R₀ and R₁ progeny followingthe method of Shure et al. (Cell 35:225-233, 1983). Fifteen microgramsof genomic DNA was digested with BglII restriction endonuclease andfractionated on a 0.8% agarose gel. The DNA was transferred to Hybond Nmembranes (Amersham) according to the standard procedure described inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1989). The membranes were probed independentlyfor the presence of genes encoding EPSPS and GOX. A 3.4 kb DNA fragmentcontaining the EPSPS gene and a 4.8 kb DNA fragment containing the GOXgene were released from pMON19574 by BglII restriction endonucleasedigestion, isolated by 0.7% agarose gel electrophoresis, and labeledwith [³²P] dCTP using a Stratagene PRIME-IT II random primer labelingkit. Probes were labeled to a specific activity of 3×10⁹ counts perminute per microgram and 1.3×10⁹ counts per minute per microgram,respectively. Membranes were hybridized for 14 hours at 42° C. in asolution containing 50% formamide, 5×SSC, 5×Denhardt's, 0.5% SDS, and100 microgram per milliliter tRNA. The condition of the final wash was0.1% SSC and 0.1% SDS at 60° C. for fifteen minutes.

EPSPS and GOX protein assays were conducted using crude protein extractsfrom leaf tissue of R₀ plants and total proteins were quantifiedfollowing the method of Bradford (Anal. Biochem. 72:248-256, 1976). Thepercentage of EPSPS and GOX protein represented in the extracts wasquantified using an ELISA method and calculated as percent totalextractable protein.

Immature embryos from the R₀ transgenic and Bobwhite control plants wereisolated twenty days after anthesis and cultured on the MMS0 medium with0.02 mM glyphosate for a germination test. Germinated and non-germinatedembryos were recorded ten days later and the data was analyzed by χ²test for 3:1 segregation. Tolerant plants from the germination test weretransferred to soil and sprayed with three milliliters per liter ofROUNDUP as described above.

Five plasmids harboring glyphosate resistance genes were used totransform immature wheat embryos as described above. pMON19338 containsa nucleotide cassette encoding a petunia EPSPS chloroplast transitpeptide in frame with an Agrobacterium strain CP4 EPSPS enzyme sequence.The nucleotide cassette is inserted downstream of a cauliflower mosaicvirus enhanced 35S promoter linked 3′ to a maize HPS70 intron sequenceand upstream of a nopaline synthase 3′ transcription termination andpolyadenylation sequence. Convenient restriction sites are positionedbetween the intron sequence and the 3′ termination sequence forinsertion of genetic elements. pMON19643 is identical to pMON19338except that a GOX enzyme encoding sequence is used in place of theAgrobacterium EPSPS enzyme encoding sequence. pMON19574 is identical topMON19338 but additionally contains a chloroplast targetedglyphosate-oxidoreductase expression cassette identical to that inpMON19643 downstream of and immediately adjacent to the EPSPS expressioncassette. pMON32570 is similar to pMON19574 in that expression cassettesencoding a chloroplast targeted EPSPS and chloroplast targeted GOX arepresent, however, an expression cassette encoding a chloroplast targetedAMPA acyltransferase enzyme is also present between the EPSPS and GOXexpression cassettes. Other elements which are present in pMON19574 andnot in the other plasmids are also worthy of mention. For example, awheat major chlorophyll a/b binding protein gene 5′ untranslated leaderis present between the enhanced 35S promoter and intron in both theEPSPS and AMPA acyltransferase expression cassettes (McElroy et al.,Plant Cell 2:163-171, 1990). Also, a wheat hsp17 gene 3′ transcriptiontermination and polyadenylation sequence is present in place of thenopaline synthase 3′ sequence for both EPSPS and AMPA acyltransferaseexpression cassettes. All plasmids produced recombinant glyphosatetolerant wheat plants using the ballistic transformation methoddescribed above. However, plasmids which were capable of expressing GOXonly or GOX along with an AMPA acyltransferase either did not producerecombinant glyphosate tolerant wheat plants or produced plants whichexperienced problems with stunted growth, aberrant segregation ofphenotypes, and infertility and were not analyzed further. The dataobtained after biolistic transformation using the described plasmids isshown in Table 18.

TABLE 18 Wheat Biolistic Transformation Data Glyphosate Tolerance #Transgenic Transformation Gene(s) # Explants Events Efficiency¹ GOX 1200 0 GOX + PhnO 434 6 1.4 EPSPS 120 6 5.0 EPSPS + GOX 120 1 0.8 EPSPS +PhnO + GOX 10,068 314 3.1 ¹transformation efficiency based on percentageof transgenic events identified from a total population of explantsarising from a combination of experiments in which a particular vectorconstruct has been bombarded into immature embryo's.

Transformed glyphosate tolerant plants arising out of thesetransformations were self crossed and allowed to produce R1 seed, whichwere used to generate R1 plants. Glyphosate tolerance generallysegregated in the expected ratio of 3:1 in R1 plants as judged by R1plant sensitivities after spraying with glyphosate at the three leafstage. Glyphosate tolerant R1 plants were self crossed and allowed toproduce R2 seed. R2 seed was germinated from a number of differentglyphosate tolerant lines to produce R2 glyphosate tolerant plants towhich [¹⁴C]-glyphosate was applied as described above. Plant leaf andstem tissues were harvested at 48 hours after glyphosate application,and water soluble compounds were extracted as described above andanalyzed by HPLC as in example 2 for the presence of [¹⁴C]-glyphosatemetabolites. The total area under the [¹⁴C] isotope labeled peakseluting from the column was summed to provide a baseline of 100%[¹⁴C]-compound identification for each sample analyzed. The results areshown in Table 19.

TABLE 19 Glyphosate Metabolism In Wheat Plant Extracts¹ Sample &Glyphosate Plant [¹⁴C]- [¹⁴C]- Acetyl- Tolerance Gene(s) Line No.Glyphosate AMPA [¹⁴C]-AMPA [¹⁴C]-Other⁴ Standard² na 30 26 31 13 na 2924 29 18 na 35 29 36 0 Growth Medium³ na 60 32 0.2 8 na 48 25 2 25 na 877 0 6 EPSPS 24756 43 25 1 31 24756 53 46 0 1 25397 61 38 0 1 25397 37 191.2 43 25397 64 20 0 16 EPSPS + PhnO + GOX 27249 6 7 85 2 27249 14 12 6113 27249 5 24 33 38 25462 48 21 0 31 25462 44 5 0 51 25462 54 35 0 1126281 48 14 17 21 26281 64 11 13 12 26281 38 7 7 48 28598 20 7 5 6828598 25 7 5 63 Bobwhite na 74 26 0 0 na 17 15 0 32 na 34 24 0 42 ¹planttissue extracts were analyzed by HPLC after [¹⁴C]-glyphosate applicationas in Example 1, and the area under the plots for each peak were summedto provide a base of 100% [¹⁴C]-compound identification for each sample.²standard solution containing approximately equal [¹⁴C] molar ratios ofeach known glyphosate metabolism related compound. ³growth mediumincluding [¹⁴C]-glyphosate; glyphosate has previously been shown to bedegraded by a photolytic process to AMPA, which can be autoacylated inthe presence of certain acyl compounds (MSL-0598). ⁴uncharacterized[¹⁴C]-labeled compounds which are resolved using the disclosedchromatographic method. Retention time of glyphosate is about 9.6minutes, AMPA is about 5.4 minutes, N-acetyl-AMPA is about 12.5 minutes,and the major [¹⁴C]-labeled impurity in the [¹⁴C]-glyphosate sample isabout 4.7 minutes.The standard solution contains approximately equal molar ratios of eachof the compounds glyphosate, AMPA and N-acetyl-AMPA, as well as a numberof impurities which are present as a result of the chemical synthesis ofthese isotope labeled compounds. Growth medium to which [¹⁴C]-glyphosatewas added was treated to the same conditions as wheat plants, ie, themedium was exposed to incident light intensities which plants received.As expected, photodegradation of glyphosate to AMPA was observed, and asmall percentage of AMPA appeared to be converted to acetyl-AMPA,probably as a result of exposure in the growth medium to other acylatedcompounds. Photodegradation of glyphosate by visible light exposure toAMPA as the major degradation product has been observed previously(Lund-Hoeie et al., Photodegradation of the herbicide glyphosate inwater. Bull. Environ. Contam. Toxicol. 36:723-729, 1986). Recombinantwheat plants transformed with an EPSPS-only plasmid did not produce[¹⁴C]-AMPA or acetyl-[¹⁴C]-AMPA from [¹⁴C]-glyphosate. [¹⁴C]-AMPA andtrace amounts of acetyl-[¹⁴C]-AMPA which were observed were within thelimits observed as a result of photodegradation in the growth mediumcontrol. Non-recombinant Bobwhite control plants treated with[¹⁴C]-glyphosate also did not produce AMPA or acetyl-AMPA. Plantstransformed with the triple gene construct plasmid containing genescapable of expressing EPSPS, PhnO and GOX produced variable results.About one third of these plants appeared to efficiently convertglyphosate to acetyl-AMPA, indicating that the GOX and PhnO enzymes werepresent and functional. Southern blot analyses demonstrated that thetransgenes were integrated into the wheat genomes and transmitted to thefollowing generations. Western blot analysis using anti-EPSPS, anti-GOX,or anti-PhnO antiserum to detect these proteins in the triple genetransformed plant extracts provided further insight into the basis forthe variable [¹⁴C]-glyphosate metabolism observation. Western blotanalysis indicated that all of the lines were producing EPSPS, howeveronly line 27249 was producing GOX and PhnO protein. This result isconsistent with the data in Table 19, which shows that line 27249efficiently metabolizes [¹⁴C]-glyphosate to acetyl-[¹⁴C]-AMPA. Thisplant line also did not demonstrate stunting, partial fertility, oraltered segregation phenotypes associated with other lines. Theseresults indicate that co-expression of GOX and AMPA acyltransferase inwheat plants expressing recombinant EPSPS provides improved herbicidetolerance.

Example 9

This example illustrates the transformation of tobacco chloroplasts witha phnO gene.

Recombinant plants can be produced in which only the mitochondrial orchloroplast DNA has been altered to incorporate the molecules envisionedin this application. Promoters which function in chloroplasts have beenknown in the art (Hanley-Bowden et al., Trends in Biochemical Sciences12:67-70, 1987). Methods and compositions for obtaining cells containingchloroplasts into which heterologous DNA has been inserted have beendescribed, for example by Daniell et al. (U.S. Pat. No. 5,693,507; 1997)and Maliga et al. (U.S. Pat. No. 5,451,513; 1995). A vector can beconstructed which contains an expression cassette from which anacyltransferase protein could be produced. A cassette could contain achloroplast operable promoter sequence driving expression of, forexample, a phnO gene, constructed in much the same manner as otherpolynucleotides herein, using PCR methodologies, restrictionendonuclease digestion, and ligation etc. A chloroplast expressible genewould provide a promoter and a 5′ untranslated region from aheterologous gene or chloroplast gene such as psbA, which would providefor transcription and translation of a DNA sequence encoding anacyltransferase protein in the chloroplast; a DNA sequence encoding anacyltransferase protein; and a transcriptional and translationaltermination region such as a 3′ inverted repeat region of a chloroplastgene that could stabilize an expressed mRNA coding for anacyltransferase protein. Expression from within the chloroplast wouldenhance gene product accumulation. A host cell containing chloroplastsor plastids can be transformed with the expression cassette and then theresulting cell containing the transformed chloroplasts can be grown toexpress the acyltransferase protein. A cassette may also include anantibiotic, herbicide tolerance, or other selectable marker gene inaddition to the acyltransferase gene. The expression cassette may beflanked by DNA sequences obtained from a chloroplast DNA which wouldfacilitate stable integration of the expression cassette into thechloroplast genome, particularly by homologous recombination.Alternatively, the expression cassette may not integrate, but byincluding an origin of replication obtained from a chloroplast DNA,would be capable of providing for replication of, for example, aheterologous phnO or other acyltransferase gene within the chloroplast.

Plants can be generated from cells containing transformed chloroplastsand can then be grown to produce seeds, from which additional plants canbe generated. Such transformation methods are advantageous over nucleargenome transformation, in particular where chloroplast transformation iseffected by integration into the chloroplast genome, because chloroplastgenes in general are maternally inherited. This provides environmentally“safer” transgenic plants, virtually eliminating the possibility ofescapes into the environment. Furthermore, chloroplasts can betransformed multiple times to produce functional chloroplast genomeswhich express multiple desired recombinant proteins, whereas nucleargenomic transformation has been shown to be rather limited when multiplegenes are desired. Segregational events are thus avoided usingchloroplast or plastid transformation. Unlike plant nuclear genomeexpression, expression in chloroplasts or plastids can be initiated fromonly one promoter and continue through a polycistronic region to producemultiple peptides from a single mRNA.

The expression cassette would be produced in much the same way thatother plant transformation vectors are constructed. Plant chloroplastoperable DNA sequences can be inserted into a bacterial plasmid andlinked to DNA sequences expressing desired gene products, such as PhnOproteins or other similar acyltransferases, so that the acyltransferaseprotein is produced within the chloroplast, obviating the requirementfor nuclear gene regulation, capping, splicing, or polyadenylation ofnuclear regulated genes, or chloroplast or plastid targeting sequences.An expression cassette comprising a phnO or similar acyltransferasegene, which is either synthetically constructed or a native gene deriveddirectly from an E. coli genome, would be inserted into a restrictionsite in a vector constructed for the purpose of chloroplast or plastidtransformation. The cassette would be flanked upstream by a chloroplastor plastid functional promoter and downstream by a chloroplast orplastid functional transcription and translation termination sequence.The resulting cassette could be incorporated into the chloroplast orplastid genome using well known homologous recombination methods.Alternatively, chloroplast or plastid transformation could be obtainedby using an autonomously replicating plasmid or other vector capable ofpropagation within the chloroplast or plastid. One means of effectuatingthis method would be to utilize a portion of the chloroplast or plastidgenome required for chloroplast or plastid replication initiation as ameans for maintaining the plasmid or vector in the transformedchloroplast or plastid. A sequence enabling stable replication of achloroplast or plastid epigenetic element could easily be identifiedfrom random cloning of a chloroplast or plastid genome into a standardbacterial vector which also contains a chloroplast or plastid selectablemarker gene, followed by transformation of chloroplasts or plastids andselection for transformed cells on an appropriate selection medium.Introduction of an expression cassette as described herein into achloroplast or plastid replicable epigenetic element would provide aneffective means for localizing an acyltransferase gene and protein tothe chloroplast or plastid.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained. Asvarious changes could be made in the above methods and compositionswithout departing from the spirit and scope of the invention, it isintended that all matter contained in the above description, and shownin the accompanying drawings and sequences, shall be interpreted asillustrative and not in a limiting sense.

REFERENCED LITERATURE

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REFERENCED PATENT DOCUMENTS

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1.-42. (canceled)
 43. A method for detoxifying a phosphonate herbicidecomprising transacetylating the phosphonate herbicide.
 44. The method ofclaim 43, wherein the phosphonate herbicide comprises a CP bond and a CNbond.
 45. The method of claim 43, wherein the phosphonate herbicide isglyphosate.